Modulation of t cells with bispecific antibodies and fc fusions

ABSTRACT

The present invention relates to methods and compositions for modulating T cells. The modulation includes suppressing or inducing regulatory T cells or cytotoxic T cells.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.14/217,166, filed Mar. 17, 2014, which claims priority to U.S.Provisional Patent Application Nos. 61/800,743, filed Mar. 15, 2013 and61/911,438, filed Dec. 3, 2013, each of which is expressly incorporatedby reference in the entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 19, 2019, isnamed 067461-5163-US01_ST25.txt and is 831,510 bytes in size.

RELATED APPLICATIONS

U.S. Ser. No. 14/216,705, filed Mar. 17, 2014; Ser. No. 13/194,904,filed Jul. 29, 2011; Ser. No. 14/205,248, filed Mar. 11, 2014; Ser. No.12/875,015, filed Sep. 2, 2010; Ser. No. 13/568,028, filed Aug. 6, 2012;Ser. No. 13/887,234, filed May 3, 2013; Ser. No. 13/648,951, filed Oct.10, 2012; 61/913,832, filed Dec. 9, 2013, and 61/938,095, filed Feb. 10,2014 are all expressly incorporated by reference in their entirety,particularly for the recitation of amino acid positions andsubstitutions, and all data, figures and legends relating thereto.

TECHNICAL FIELD

The present disclosure relates to methods and compositions formodulating T cells.

BACKGROUND OF THE INVENTION

Immune system homeostasis relies on a fine balance between a variety ofT cell populations, including effector CD8 and CD4 T cells andregulatory T cells. In disease states however, such as cancer andautoimmune disease, this balance can be perturbed. In cancer,infiltrating anti-tumor cytotoxic T cells can be prevented fromattacking cancer cells by tumor-resident regulatory T cells. This can beseen from analysis of most human tumors, in which there is a significantcorrelation between immune infiltration by cytotoxic T cells andimproved outcome, whereas infiltration by regulatory T cells is insteadassociated with a poor outcome. Indeed, several studies havedemonstrated prognostic significance of the CD8/Treg tumor ratio.Numerous mouse models have shown that depletion of Treg with anti-CD25antibody before tumor implantation can have a dramatic impact onprevention of tumor growth. In autoimmune diseases, effector T cellsremain unregulated and attack the body's own tissues. A major premise inthis regard is that defects in Treg cell number or function are acontributing factor. Therefore, the ability to alter the balance betweencytotoxicity and regulation by fine-tuning the T cell response has greatpotential for the treatment of cancer, autoimmune, and other diseases.

One approach to controlling the balance of effector to regulatory Tcells is to target the Treg population for direct modulation. However,despite years of effort, the discovery of a single Treg-specific surfacemarker has been elusive, frustrating efforts to deplete themspecifically with monoclonal antibodies.

Effector versus regulatory T cells can be loosely identified by theirsurface markers, which can change based on their activation state.Cytotoxic T cells express CD8, which interacts with class I MHC.Effector helper T cells express CD4, which interacts with class II MHCon antigen-presenting cells. The hallmark of Treg cells is constitutiveexpression of both CD4 and CD25. CD25 is the alpha component of the IL2receptor (IL2Rα), which, together with CD122 (IL2Rβ) and the commoncytokine receptor γ-chain(y_(c)) (CD132) form the trimeric high-affinityreceptor for IL2. Several approaches have attempted Treg-specificdepletion by targeting the high-affinity IL2 receptor CD25 (IL2Rα) withanti-CD25 antibodies such as daclizumab, or with IL2-diptheria toxin(IL2-DT) fusions. However, CD25 alone is not an ideal target because italso expressed on CD8 and CD4 effector T cells upon activation. Thus,approaches that target Treg CD25 by itself might defeat their ownpurpose by also depleting the activated effector cells that couldpotentially attack the tumor.

Because of the importance of IL2 for T cell proliferation andhomeostasis, a variety of approaches to T cell modulation have utilizedIL2 itself or blocking of its high-affinity receptor component CD25.Systemic IL2 administration (Proleukin) is an approved therapy formetastatic melanoma and metastatic renal cell carcinoma based on itsability to promote expansion of effector T cells. However, systemic IL2administration is also expected to promote the suppressive Tregpopulation, potentially diminishing or confounding the desiredenhancement of cytotoxic T cells. Furthermore, systemic IL2administration is also associated with a variety of toxicities. Patientsreceiving systemic IL2 treatment frequently experience severecardiovascular, pulmonary, renal, hepatic, gastrointestinal,neurological, cutaneous, haematological and systemic adverse events. Themajority of these side effects can be explained by the development ofso-called vascular leak syndrome (VLS), a pathological increase invascular permeability leading to pulmonary edema and other issues. Thereis no treatment of VLS other than withdrawal of IL2. These problems haveled to the pursuit of IL2 variants that perturb its affinity for one ormore of its receptor subunits. Alternatively, anti-CD25 antibodies thatblock IL2-mediated T cell expansion have been utilized to treat variousdiseases. Zenapax (daclizumab) is an approved immunosuppressant fororgan transplantation and is being investigated for the treatment ofautoimmune diseases such as multiple sclerosis. These uses weredeveloped based on daclizumab's presumed ability to reduce effector Tcell responses. However, due to the more recently recognized dependenceof Treg on IL2 for survival, daclizumab is now—somewhatparadoxically—being utilized in efforts to reduce Treg numbers inoncology. Because of the strong potential of either IL2 or anti-CD25agents to promote or reduce both effector T cells and Treg with limitedselectivity, there is a strong need in the field to create moreselective Treg modulators.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods and compositions forsuppressing and inducing T cells. In preferred aspects, the methods andcompositions of the invention suppress or induce specific T cell typeswith little or no impact on other T cell types. In further embodiments,the methods and compositions of the invention suppress or induceregulatory T cells with little or no impact on other T cell types,including cytotoxic T cells. In other embodiments, the methods andcompositions of the invention suppress or induce cytotoxic T cells withlittle or no impact on other T cell types, including regulatory T cells.

In one aspect, the present invention provides a method for suppressing Tcells that includes the step of administering a composition comprising abispecific antibody, wherein that bispecific antibody includes: (a) afirst monomer that has (i) a first heavy chain constant region with afirst variant Fc domain; and (ii) an anti-CD25 binding moiety; and (b) asecond monomer that has (i) a second heavy chain constant region with asecond variant Fc domain; and (ii) a member selected from the group: ananti-CD4 binding moiety, an anti-CD8 binding moiety, an anti-CCR4moiety, an anti-GITR binding moiety, and an anti-PD-1 binding moiety. Inspecific embodiments, the first variant Fc domain has a different aminoacid sequence than the second variant Fc domain. The administration ofsuch a bispecific antibody serves to suppress the T cells. Suppressioncan be measured using assays known in the art, including cellproliferation assays. Suppression can be shown in such assays by adecrease in cell proliferation and/or general T cell number as comparedto the proliferation and/or numbers seen in the absence of thebispecific antibody of the invention.

In further embodiments and in accordance with the above, the T cellssuppressed by the methods of the invention are regulatory T cells. Instill further embodiments, the second monomer comprises the anti-CD4binding moiety, and the bispecific antibody specifically targetsregulatory T cells with limited to no impact on other T cell types.

In still further embodiments and in accordance with any of the above,the anti-CD25 binding moiety is an anti-CD25 scFV sequence that iscovalently attached to the first heavy chain sequence.

In still further embodiments and in accordance with any of the above,the T cells suppressed by the methods and compositions of the inventionare cytotoxic T-cells. In yet further embodiments, the second monomercomprises said anti-CD8 binding moiety, and the bispecific antibodyspecifically targets cytotoxic T cells with limited to no impact onother T cell types. In yet further embodiments, the anti-CD8 bindingmoiety comprises all or a portion of an antigen binding region of anantibody selected from the group consisting of MCD8, 3B5, Sk1, OKT-8,and DK-25.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domains include a set of amino acidsubstitutions selected from those sets depicted in FIG. 33A-33C.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domains comprise a set of amino acidsubstitutions selected from the group consisting of those sets depictedin FIG. 34A-34C.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domains comprise a set of amino acidsubstitutions selected from the group consisting of those sets depictedin FIG. 35.

In still further embodiments and in accordance with any of the above,the first and/or second variant Fc domain comprises an amino acidvariant selected from the group consisting of: 236R, 239D, 239E, 243L,M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E,M428L, N434A, N434S, 236R/328R, 239D/332E, M428L, 236R/328F,V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L, Y436V/M428L,Y436I/N434S, Y436V/N434S, 239D/332E/330L, M252Y/S254T/T256E,V259I/V308F/M428L, and E233P/L234V/L235A/G236del/S267K.

In still further embodiments and in accordance with any of the above,the bispecific antibody comprises a sequence selected from the sequencesdepicted in FIGS. 30-31.

In still further embodiments and in accordance with any of the above,the first monomer comprises a sequence according to the sequencedesignated as11209-OKT4A_H0L0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_IgG1,Heavy chain 2 (Heavy chain 2(Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(+RR)) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as11209-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_IgG1,Heavy chain 1 (OKT4A_HOL0_scFv_GDQ-Fc(216)_IgG1_pI_ISO(−)) in FIG.30A-30FF.

In still further embodiments and in accordance with any of the above,the first monomer comprises a sequence according to the sequencedesignated as12143-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_C220S/FcKO,Heavy chain 2 (Anti-TAC_H1L1_scFv_Fc(216)_IgG1_pI_ISO(+RR)_G236R/L328R)in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as12143-OKT4A_H0L0_scFv_Anti-TAC_H1L1_scFv_-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_C220S/FcKO,Heavy chain 1 (OKT4A_HOL0_scFv_Fc(216)_IgG1_pI_ISO(−)_G236R/L328R) inFIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the first monomer comprises a sequence according to the sequencedesignated as13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(−)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Heavy chain 2(Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG.30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(−)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Heavy chain 1(OKT4A_H1_IgG1_pI_ISO(−)_G236R/L328R) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer further comprises a sequence according to thesequence designated as13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(−)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Light chain (OKT4A_L1) in FIG.30A-30FF.

In a further aspect, the present invention provides a method forstimulating T cells that includes administering a heterodimeric protein,where the heterodimeric protein includes: (a) a first monomer with (i) afirst heavy chain constant region comprising a first variant Fc domain;(ii) an IL-2 protein; and (b) a second monomer with: (i) a second heavychain constant region comprising a second variant Fc domain; (ii) amember selected from the group consisting of: an anti-CD4 bindingmoiety, an anti-CD8 binding moiety, an anti-CTLA4 binding moiety, ananti-CCR4 binding moiety, an anti-PD-1 binding moiety, and an anti-GITRbinding moiety. In further embodiments, the first variant Fc domain hasa different amino acid sequence than the second variant Fc domain.Administration of this heterodimeric protein stimulates the T cells. Aswill be appreciated, the IL2 protein may comprise a full length proteinor a portion of the full length IL2 protein. In further embodiments, thefull or portion of the IL2 protein that is part of the heterodimericprotein comprises a human IL2 protein sequence.

In a further embodiment and in accordance with the above, the T cellsare regulatory T cells and the second monomer is the anti-CD4 bindingmoiety.

In still further embodiments and in accordance with any of the above,the second monomer further includes: (a) the second heavy chain constantregion further having a heavy chain variable domain, and (b) a lightchain sequence, where the heavy chain variable domain and the lightchain sequence together form antigen binding moiety, including withoutlimitation the anti-CD4 binding moiety.

In still further embodiments and in accordance with any of the above,the stimulated T cells are cytotoxic T cells and the second monomercomprises the anti-CD8 binding moiety. In yet further embodiments, theanti-CD8 binding moiety comprises all or a portion of an antigen bindingregion of an antibody selected from the group consisting of MCD8, 3B5,Sk1, OKT-8, and DK-25.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domains include a set of amino acidsubstitutions selected from the group consisting of those sets depictedin FIG. 33, 34 or 35.

In still further embodiments and in accordance with any of the above,the first and/or second variant Fc domain comprises an amino acidvariant selected from the group consisting of: 236R, 239D, 239E, 243L,M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E,M428L, N434A, N434S, 236R/328R, 239D/332E, M428L, 236R/328F,V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L, Y436V/M428L,Y436I/N434S, Y436V/N434S, 239D/332E/330L, M252Y/S254T/T256E,V259I/V308F/M428L, and E233P/L234V/L235A/G236del/S267K.

In still further embodiments and in accordance with any of the above,the heterodimeric protein comprises a sequence selected from thesequences depicted in FIGS. 30-31.

In still further embodiments and in accordance with any of the above,the first monomer comprises a sequence according to the sequencedesignated as13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(−/+RR)_C220S_G236R/L328R, Heavy chain1 (hIL2_IgG1_pI_ISO(−)_C220S/G236R/L328R) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(−/+RR)_C220S_G236R/L328R, Heavy chain2 (OKT4A_H1_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer further comprises a sequence according to thesequence designated as13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(−/+RR)_C220S_G236R/L328R, Light chain(OKT4A_L1) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as 13038-OKT4A_H1L1_IgG1_G236R/L328R_hIL2(2), Heavy chain(OKT4A_H1_IgG1_G236R/L328R_hIL2) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer further comprises a sequence according to thesequence designated as 13038-OKT4A_H1L1_IgG1_G236R/L328R_hIL2(2), Lightchain (OKT4A_L1) in FIG. 30A-30FF.

In further aspects and in accordance with any of the above, the presentinvention provides a composition comprising a heterodimeric antibody,where the heterodimeric antibody includes: (a) a first monomer having(i) a first antigen-binding domain, which is an anti-CD25 bindingdomain; (ii) a first heavy chain sequence comprising a first variant Fcdomain as compared to a human Fc domain; and (b) a second monomer having(i) a second antigen-binding domain that binds to a member selected fromthe group consisting of: CD4, CD8, CCR4, GITR, and PD-1, and (ii) asecond heavy chain sequence comprising a second variant Fc domain ascompared to a human Fc domain. In further embodiments, the first andsecond variant Fc domains have different amino acid sequences.

In further embodiments and in accordance with the above, theantigen-binding domain binds to CD4.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domain includes an amino acid variantindependently selected from the variants listed in FIG. 33, 34, or 35.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domain comprises an amino acid variantselected from the group consisting of: L368D/K370S and S364K;L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S andS364K/E357Q; and K370S and S364K/E357Q.

In still further embodiments and in accordance with any of the above,the first and/or second variant Fc domain further comprises an aminoacid variant selected from the group consisting of: 236R, 239D, 239E,243L, M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D,332E, M428L, N434A, N434S, 236R/328R, 239D/332E, M428L, 236R/328F,V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L, Y436V/M428L,Y436I/N434S, Y436V/N434S, 239D/332E/330L, M252Y/S254T/T256E,V259I/V308F/M428L, and E233P/L234V/L235A/G236del/S267K.

In still further embodiments and in accordance with any of the above,the anti-CD25 binding domain is an anti-CD25 scFv sequence and iscovalently attached to said first heavy chain sequence.

In still further embodiments and in accordance with any of the above,the second antigen-binding domain comprises an scFv sequence.

In still further embodiments and in accordance with any of the above,the second monomer further has the second heavy chain sequence furthercomprising a heavy chain variable domain, and a light chain sequence,where the heavy chain variable domain and the light chain sequence formsaid second antigen-binding domain.

In still further embodiments and in accordance with any of the above,the composition comprises a format in accordance with a format asdepicted in FIG. 3 or FIGS. 36A-37U.

In a further aspect, the present invention provides a compositioncomprising a heterodimeric protein that has: (a) a first monomercomprising: (i) a first protein comprising a cell marker; (ii) a firstheavy chain sequence with a first variant Fc domain as compared to ahuman Fc domain; and (b) a second monomer comprising: (i) anantigen-binding domain that binds to a member selected from the groupconsisting of: CD4, CD8, CTLA-4, CCR4, and PD-1, and (ii) a second heavychain sequence comprising a second variant Fc domain as compared to ahuman Fc domain. In further embodiments, the first and second variant Fcdomains have different amino acid sequences.

In still further embodiments and in accordance with any of the above,the protein of the first monomer comprises a regulatory T cell markerselected from the group listed in FIG. 32. In other embodiments, theprotein of the first monomer comprises a cytokine. In yet furtherembodiments, the cytokine is IL2.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domain comprises an amino acid variantindependently selected from the variants listed in FIG. 33, 34 or 35.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domain includes an amino acid variantindependently selected from the group consisting of: L368D/K370S andS364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S andS364K/E357Q; and K370S and S364K/E357Q.

In still further embodiments and in accordance with any of the above,the first and/or second variant Fc domain further includes an amino acidvariant selected from the group consisting of: 236R, 239D, 239E, 243L,M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E,M428L, N434A, N434S, 236R/328R, 239D/332E, M428L, 236R/328F,V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L, Y436V/M428L,Y436I/N434S, Y436V/N434S, 239D/332E/330L, M252Y/S254T/T256E,V259I/V308F/M428L, and E233P/L234V/L235A/G236del/S267K.

In still further embodiments and in accordance with any of the above,the antigen-binding domain is an scFv sequence that is covalentlyattached to said second heavy chain sequence.

In still further embodiments and in accordance with any of the above,the second monomer further includes: (a) the second heavy chain sequencefurther comprising a heavy chain variable domain, and (b) a light chainsequence, wherein said heavy chain variable domain and said light chainsequence form said second antigen-binding domain.

In still further embodiments and in accordance with any of the above,the heterodimeric protein comprises a sequence as listed in FIGS. 30 and31.

In still further embodiments and in accordance with any of the above,the invention provides one or more nucleic acids encoding a compositionaccording to any of the compositions described above. In yet furtherembodiments, the invention includes a host cell expressing those one ormore nucleic acids. In yet further embodiments, the present inventionprovides a method of making any of the compositions described herein,the method including the step of culturing a host cell or more nucleicacids encoding a composition according to any of the compositionsdescribed above under conditions whereby the composition is produced.

In further aspects, the present invention provides a method of purifyinga heterodimeric protein or bispecific antibody in accordance with any ofthe above, the method including: (a) providing a composition inaccordance with any of the above, (b) loading the composition onto anion exchange column; and (c) collecting a fraction containing theheterodimeric protein or bispecific antibody, thus purifying the proteinor antibody.

In a further aspect, the present invention provides a method of treatingcancer in a subject, the method comprising administering to said subjecta composition comprising a bispecific antibody, where the bispecificantibody includes: (a) a first monomer that has (i) a first heavy chainconstant region with a first variant Fc domain; and (ii) an anti-CD25binding moiety; and (b) a second monomer that has (i) a second heavychain constant region with a second variant Fc domain; and (ii) a memberselected from the group: an anti-CD4 binding moiety, an anti-CD8 bindingmoiety, an anti-CCR4 moiety, an anti-GITR binding moiety, and ananti-PD-1 binding moiety. In specific embodiments, the first variant Fcdomain has a different amino acid sequence than the second variant Fcdomain. The administration of such a bispecific antibody serves tosuppress the T cells. Suppression can be measured using assays known inthe art, including cell proliferation assays. Suppression can be shownin such assays by a decrease in cell proliferation and/or general T cellnumber as compared to the proliferation and/or numbers seen in theabsence of the bispecific antibody of the invention.

In further embodiments and in accordance with the above, the T cellssuppressed by the methods of the invention are regulatory T cells. Instill further embodiments, the second monomer comprises the anti-CD4binding moiety, and the bispecific antibody specifically targetsregulatory T cells with limited to no impact on other T cell types.

In still further embodiments and in accordance with any of the above,the anti-CD25 binding moiety is an anti-CD25 scFV sequence that iscovalently attached to the first heavy chain sequence.

In still further embodiments and in accordance with any of the above,the T cells suppressed by the methods and compositions of the inventionare cytotoxic T-cells. In yet further embodiments, the second monomercomprises said anti-CD8 binding moiety, and the bispecific antibodyspecifically targets cytotoxic T cells with limited to no impact onother T cell types. In yet further embodiments, the anti-CD8 bindingmoiety comprises all or a portion of an antigen binding region of anantibody selected from the group consisting of MCD8, 3B5, Sk1, OKT-8,and DK-25.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domains include a set of amino acidsubstitutions selected from those sets depicted in FIG. 33.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domains comprise a set of amino acidsubstitutions selected from the group consisting of those sets depictedin FIG. 34A-34C.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domains comprise a set of amino acidsubstitutions selected from the group consisting of those sets depictedin FIG. 35.

In still further embodiments and in accordance with any of the above,the first and/or second variant Fc domain comprises an amino acidvariant selected from the group consisting of: 236R, 239D, 239E, 243L,M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E,M428L, N434A, N434S, 236R/328R, 239D/332E, M428L, 236R/328F,V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L, Y436V/M428L,Y436I/N434S, Y436V/N434S, 239D/332E/330L, M252Y/S254T/T256E,V259I/V308F/M428L, and E233P/L234V/L235A/G236del/S267K.

In still further embodiments and in accordance with any of the above,the bispecific antibody comprises a sequence selected from the sequencesdepicted in FIGS. 30-31.

In still further embodiments and in accordance with any of the above,the first monomer comprises a sequence according to the sequencedesignated as11209-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_IgG1,Heavy chain 2 (Heavy chain 2(Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(+RR)) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as11209-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_IgG1,Heavy chain 1 (OKT4A_HOL0_scFv_GDQ-Fc(216)_IgG1_pI_ISO(−)) in FIG.30A-30FF.

In still further embodiments and in accordance with any of the above,the first monomer comprises a sequence according to the sequencedesignated as12143-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_C220S/FcKO,Heavy chain 2 (Anti-TAC_H1L1_scFv_Fc(216)_IgG1_pI_ISO(+RR)_G236R/L328R)in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as12143-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_C220S/FcKO,Heavy chain 1 (OKT4A_HOL0_scFv_Fc(216)_IgG1_pI_ISO(−)_G236R/L328R) inFIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the first monomer comprises a sequence according to the sequencedesignated as13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(−)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Heavy chain 2(Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG.30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(−)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Heavy chain 1(OKT4A_H1_IgG1_pI_ISO(−)_G236R/L328R) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer further comprises a sequence according to thesequence designated as13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(−)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Light chain (OKT4A_L1) in FIG.30A-30FF.

In a further aspect, the present invention provides a method fortreating autoimmune disease in a subject that includes administering aheterodimeric protein to the subject, where the heterodimeric proteinincludes: (a) a first monomer with (i) a first heavy chain constantregion comprising a first variant Fc domain; (ii) an IL-2 protein; and(b) a second monomer with: (i) a second heavy chain constant regioncomprising a second variant Fc domain; (ii) a member selected from thegroup consisting of: an anti-CD4 binding moiety, an anti-CD8 bindingmoiety, an anti-CTLA4 binding moiety, an anti-CCR4 binding moiety, ananti-PD-1 binding moiety, and an anti-GITR binding moiety. In furtherembodiments, the first variant Fc domain has a different amino acidsequence than the second variant Fc domain. Administration of thisheterodimeric protein stimulates the T cells. As will be appreciated,the IL2 protein may comprise a full length protein or a portion of thefull length IL2 protein. In further embodiments, the full or portion ofthe IL2 protein that is part of the heterodimeric protein comprises ahuman IL2 protein sequence.

In a further embodiment and in accordance with the above, the T cellsare regulatory T cells and the second monomer is the anti-CD4 bindingmoiety.

In still further embodiments and in accordance with any of the above,the second monomer further includes: (a) the second heavy chain constantregion further having a heavy chain variable domain, and (b) a lightchain sequence, where the heavy chain variable domain and the lightchain sequence together form antigen binding moiety, including withoutlimitation the anti-CD4 binding moiety.

In still further embodiments and in accordance with any of the above,the stimulated T cells are cytotoxic T cells and the second monomercomprises the anti-CD8 binding moiety. In yet further embodiments, theanti-CD8 binding moiety comprises all or a portion of an antigen bindingregion of an antibody selected from the group consisting of MCD8, 3B5,Sk1, OKT-8, and DK-25.

In still further embodiments and in accordance with any of the above,the first and second variant Fc domains include a set of amino acidsubstitutions selected from the group consisting of those sets depictedin FIG. 33, 34 or 35.

In still further embodiments and in accordance with any of the above,the first and/or second variant Fc domain comprises an amino acidvariant selected from the group consisting of: 236R, 239D, 239E, 243L,M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E,M428L, N434A, N434S, 236R/328R, 239D/332E, M428L, 236R/328F,V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L, Y436V/M428L,Y436I/N434S, Y436V/N434S, 239D/332E/330L, M252Y/S254T/T256E,V259I/V308F/M428L, and E233P/L234V/L235A/G236del/S267K.

In still further embodiments and in accordance with any of the above,the heterodimeric protein comprise a sequence selected from thesequences depicted in FIGS. 30A-31OOO.

In still further embodiments and in accordance with any of the above,the first monomer comprises a sequence according to the sequencedesignated as13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(−/+RR)_C220S_G236R/L328R, Heavy chain1 (hIL2_IgG1_pI_ISO(−)_C220S/G236R/L328R) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(−/+RR)_C220S_G236R/L328R, Heavy chain2 (OKT4A_H1_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer further comprises a sequence according to thesequence designated as13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(−/+RR)_C220S_G236R/L328R, Light chain(OKT4A_L1) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer comprises a sequence according to the sequencedesignated as 13038-OKT4A_H1L1_IgG1_G236R/L328R_hIL2(2), Heavy chain(OKT4A_H1_IgG1_G236R/L328R_hIL2) in FIG. 30A-30FF.

In still further embodiments and in accordance with any of the above,the second monomer further comprises a sequence according to thesequence designated as 13038-OKT4A_H1L1_IgG1_G236R/L328R_hIL2(2), Lightchain (OKT4A_L1) in FIG. 30A-30FF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Diagram illustrating suppression of Treg cells withanti-CD4×anti-CD25 bispecifics.

FIG. 2. Evaluation of the ability of various anti-CD25 heavy chains topair with anti-CD4 light chains and anti-CD4 heavy chains to pair withanti-CD25 light chains. Biacore was used to examine binding of thevarious pairs to both CD4 and CD25 and the results tabulated. TheHuMax-TAC anti-CD25 heavy chain has the unique ability to pair with theanti-CD4 lights chains of OKT4A and zanolimumab, with theHuMax-TAC/OKT4A pair showing the strongest binding.

FIG. 3. Diagram showing three exemplary anti-CD4×anti-CD25 bispecificformats. Common light-chain, dual scFv, and Fab/scFv-Fc formats areshown. Purification of heterodimer formats is accomplished utilizingProtein A and IEX chromatography. IgG1, FcR enhanced, and/or FcRknockout Fc regions may be used in further embodiments of the invention.

FIG. 4. Dual scFv-Fc bispecific antibodyOKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(−)/pI_ISO(+RR)_IgG1_S239D/1332Ecan bind to CD25 and CD4 simultaneously. The antibody was bound to aCD25 chip on Biacore followed by binding of CD4. As a control, anti-CD25antibody Anti-TAC_H1L1_IgG1 does not bind CD4.

FIG. 5. Suppression of Treg cells by anti-CD4×anti-CD25 bispecifics.Proliferation of Tregs was assayed using CFSE in the presence ofbispecific or control antibodies with 15 U/mL IL2.

FIG. 6. Effect of anti-CD4×anti-CD25 bispecifics on helper (CD4⁺CD25⁺)and cytotoxic (CD8⁺CD25⁺) T cell populations.

FIG. 7. Effect of bispecific antibodies and control anti-CD25 andanti-CD4 antibodies on cell proliferation of Tregs, CD4+ T-effectors,and CD8+ T-effectors. Bispecific antibody 12143 has higher potency onTregs compared to controls as well as lower potency compared to controlson unwanted suppression of T effector cells.

FIG. 8. Direct binding of anti-CD4×anti-CD25 bispecifics and controlantibodies on Treg cells.

FIG. 9. Direct binding of anti-CD4×anti-CD25 bispecifics and controlantibodies on Treg cells. Variants engineered for altered CD25 affinityare shown.

FIG. 10. Direct binding of anti-CD4×anti-CD25 bispecifics and controlantibodies on Treg cells. Variants containing the ibalizumab anti-CD4 Fvare shown.

FIG. 11. Direct binding of anti-CD4×anti-CD25 bispecifics and controlantibodies on Treg cells.

FIG. 12. Suppression of Treg cells by anti-CD4×anti-CD25 bispecifics.Proliferation of Tregs was assayed using CFSE in the presence ofbispecific or control antibodies with 15 U/mL IL2.

FIG. 13. Suppression of Treg cells by anti-CD4×anti-CD25 bispecifics.Proliferation of Tregs was assayed using Alamar Blue in the presence ofbispecific or control antibodies with 15 U/mL IL2.

FIG. 14. Binding of anti-CD4×anti-CD25 bispecifics and controlantibodies to purified naïve human CD4+ T cells.

FIG. 15. Effect of altering CD25 binding affinity on suppression of Tregcells by anti-CD4×anti-CD25 bispecifics. Proliferation of Tregs wasassayed using CFSE in the presence of bispecific or control antibodieswith 15 U/mL IL2.

FIG. 16. Direct binding of altered CD25 affinity anti-CD4×anti-CD25bispecifics to purified naïve human CD4+ T cells.

FIG. 17. Direct binding of anti-CD4×anti-CD25 bispecifics and controlsto activated T effector cells (CD4+CD25+).

FIG. 18. Direct binding of altered CD25 affinity anti-CD4×anti-CD25bispecifics to activated T effector cells (CD4+CD25+).

FIG. 19. Direct binding of anti-CD4×anti-CD25 bispecifics and controlsto activated T effector cells (CD4+CD25+).

FIG. 20. Direct binding of anti-CD4×anti-CD25 bispecifics and controlsto activated T effector cells (CD8+CD25+).

FIG. 21. Direct binding of altered CD25 affinity anti-CD4×anti-CD25bispecifics to activated T effector cells (CD8+CD25+).

FIG. 22. Direct binding of anti-CD4×anti-CD25 bispecifics and controlsto activated T effector cells (CD8+CD25+).

FIG. 23. Summary of IL2 variants that can be used for suppression orinduction of Tregs.

FIG. 24. Diagram illustrating induction of Treg cells with anti-CD4×IL2Fc-fusions. An example construct is also shown.

FIG. 25. Exemplary IL2 Fc-fusions and bispecific antibody-IL2 Fc-fusionsfor induction of Tregs.

FIG. 26. Purification and analysis of anti-CD4×IL2 Fc-fusions.Fc-fusions are purified by Protein A and IEX chromatography, and purityassessed by SEC and SDS-PAGE.

FIG. 27. Induction of regulatory T cells (Tregs) by anti-CD4×IL2Fc-fusions. Induction of Tregs was assayed using the alamar blue cellviability assay in the presence of anti-CD4×IL2 Fc-fusions or controlantibodies.

FIG. 28. Diagram illustrating suppression of activated cytotoxic(CD8+CD25+) T cells by anti-CD8×anti-CD25 bispecific antibodies.

FIG. 29. Diagram illustrating induction of naïve and activated cytotoxic(CD8⁺CD25⁺) T cells by anti-CD8×IL2 Fc-fusions.

FIG. 30A-30FF. Sequences of anti-CD4×anti-CD25 bispecifics, anti-CD4×IL2Fc-fusions, and control antibodies.

FIG. 31A-31OOO. Sequences of T cell modulating bispecifics, Fc-fusions,and control antibodies.

FIG. 32. Table of exemplary Treg markers for use in embodiments of theinvention.

FIG. 33A-33C. Table of exemplary amino acid variants for embodiments ofheterodimeric proteins of the invention.

FIG. 34A-34C. Table of exemplary amino acid variants for embodiments ofheterodimeric proteins of the invention.

FIG. 35. Table of exemplary amino acid variants for embodiments ofheterodimeric proteins of the invention.

FIG. 36A-36M. Illustration of a number of heterodimeric protein formats,including heterodimeric Fc fusion proteins as well as heterodimericantibodies. FIG. 36A shows the basic concept of a dimeric Fc region withfour possible fusion partners A, B, C and D. A, B, C and D areoptionally and independently selected from immunoglobulin domain(s)(e.g. Fab, vH, vL, scFv, scFv2, scFab, dAb, etc.), peptide(s), cytokines(e.g. IL-2, IL-10, IL-12, GCSF, GM-CSF, etc.), chemokine(s) (e.g.RANTES, CXCL9, CXCL10, CXCL12, etc.), hormone(s) (e.g. FSH, growthhormone), immune receptor(s) (e.g. CTLA-4, TNFR1, TNFRII, other TNFSF,other TNFRSF, etc.) and blood factor(s) (e.g. Factor VII, Factor VIII,Factor IX, etc.). Domains filled with solid white or solid black areengineered with heterodimerization variants as outlined herein. FIG. 36Bdepicts the “triple F” format (sometimes also referred to as the“bottle-opener” configuration as discussed below). FIG. 36C shows a“triple F” configuration with another scFv attached to the Fab monomer(this one, along with FIG. 36F, has a greater molecular weightdifferential as well). FIG. 36D depicts a “triple F” with another scFvattached to the scFv monomer. FIG. 36E depicts a “three scFv” format.FIG. 36F depicts an additional Fab attached to the Fab monomer. FIG. 36Gdepicts a Fab hooked to one of the scFv monomers. FIGS. 1H-1L showadditional varieties of “higher multispecificity” embodiments of the“triple F” format, all with one monomer comprising an scFv (and all ofwhich have molecular weight differentials which can be exploited forpurification of the heterodimers). FIG. 36H shows a “Fab-Fv” format withbinding to two different antigens, with FIG. 361 depicting the “Fab-Fv”format with binding to a single antigen (e.g. bivalent binding toantigen 1). FIGS. 36J and 36K depicts a “Fv-Fab” format with similarbivalent or monovalent additional antigen binding. FIG. 36L depicts onemonomer with a CH1-CL attached to the second scFv. FIG. 36M depicts adual scFv format.

FIG. 37A-37U. Depicts a wide variety of the multispecific (e.g.heterodimerization) formats and the combinations of different types ofheterodimerization variants that can be used in the present invention(these are sometimes referred to herein as “heterodimeric scaffolds”).Note in addition that all of these formats can include addition variantsin the Fc region, as more fully discussed below, including “ablation” or“knock out” variants (FIG. 39), Fc variants to alter FcγR binding(FcγRIIb, FcγRIIIa, etc.), Fc variants to alter binding to FcRnreceptor, etc. FIG. 37A shows a dual scFv-Fc format, that, as for allheterodimerization formats herein can include heterodimerizationvariants such as pI variants, knobs in holes (KIH, also referred toherein as steric variants or “skew” variants), charge pairs (a subset ofsteric variants), isosteric variants, and SEED body (“strand-exchangeengineered domain”; see Klein et al., mAbs 4:6 653-663 (2012) and Daviset al, Protein Eng Des Sel 2010 23:195-202) which rely on the fact thatthe CH3 domains of human IgG and IgA do not bind to each other. FIG. 37Bdepicts a bispecific IgG, again with the option of a variety ofheterodimerization variants. FIG. 37C depicts the “one armed” version ofDVD-Ig which utilizes two different variable heavy and variable lightdomains. FIG. 37D is similar, except that rather than an “empty arm”,the variable heavy and light chains are on opposite heavy chains. FIG.37E is generally referred to as “mAb-Fv”. FIG. 37F depicts a multi-scFvformat; as will be appreciated by those in the art, similar to the “A,B, C, D” formats discussed herein, there may be any number of associatedscFvs (or, for that matter, any other binding ligands orfunctionalities). Thus, FIG. 37F could have 1, 2, 3 or 4 scFvs (e.g. forbispecifics, the scFv could be “cis” or “trans”, or both on one “end” ofthe molecule). FIG. 37G depicts a heterodimeric FabFc with the Fab beingformed by two different heavy chains one containing heavy chain Fabsequences and the other containing light chain Fab sequences. FIG. 37Hdepicts the “one armed Fab-Fc”, where one heavy chain comprises the Fab.FIG. 37I depicts a “one armed scFv-Fc”, wherein one heavy chain Fccomprises an scFv and the other heavy chain is “empty”. FIG. 37J shows ascFv-CH3, wherein only heavy chain CH3 regions are used, each with theirown scFv. FIG. 37K depicts a mAb-scFv, wherein one end of the moleculeengages an antigen bivalently with a monovalent engagement using an scFvon one of the heavy chains. FIG. 37L depicts the same structure exceptthat both heavy chains comprise an additional scFv, which can eitherbind the same antigen or different antigens. FIG. 37M shows the“CrossMab” structure, where the problem of multiplex formation due totwo different light chains is addressed by switching sequences in theFab portion. FIG. 37N depicts an scFv, FIG. 370 is a “BITE” or scFv-scFvlinked by a linker as outlined herein, FIG. 37P depicts a DART, FIG. 37Qdepicts a TandAb, and FIG. 37R shows a diabody. FIGS. 37S, 37T and 37Udepict additional alternative scaffold formats that find use in thepresent invention.

FIG. 38. Depicts a list of isotypic and isosteric variant antibodyconstant regions and their respective substitutions. pI_(−) indicateslower pI variants, while pI_(+) indicates higher pI variants. These canbe optionally and independently combined with other heterodimerizationvariants of the invention.

FIG. 39. Depicts a number of suitable “knock out” (“KO”) variants toreduce binding to some or all of the FcγR receptors. As is true for manyif not all variants herein, these KO variants can be independently andoptionally combined, both within the set described in FIG. 39 and withany heterodimerization variants outlined herein, including steric and pIvariants. For example, E233P/L234V/L235A/G236del can be combined withany other single or double variant from the list. In addition, while itis preferred in some embodiments that both monomers contain the same KOvariants, it is possible to combine different KO variants on differentmonomers, as well as have only one monomer comprise the KO variant(s).Reference is also made to the Figures and Legends of U.S. Ser. No.61/913,870, all of which is expressly incorporated by reference in itsentirety as it relates to “knock out” or “ablation” variants.

FIG. 40. Depicts a number of charged scFv linkers that find use inincreasing or decreasing the pI of heterodimeric proteins that utilizeone or more scFv as a component. A single prior art scFv linker with asingle charge is referenced as “Whitlow”, from Whitlow et al., ProteinEngineering 6(8):989-995 (1993). It should be noted that this linker wasused for reducing aggregation and enhancing proteolytic stability inscFvs.

FIG. 41A-41B. FIGS. 41A and 41B provides an additional list of potentialheterodimerization variants for use in the present invention, includingisotypic variants.

FIG. 42A-42J. Depicts additional exemplary heterodimerization variantpairs for use in heterodimeric proteins of the invention.

FIG. 43. Depicts amino acid sequences of wild-type constant regions usedin the invention.

FIG. 44. Depicts two different Triple F embodiments for bispecificantibodies of the invention.

FIG. 45. Literature pIs of the 20 amino acids. It should be noted thatthe listed pIs are calculated as free amino acids; the actual pI of anyside chain in the context of a protein is different, and thus this listis used to show pI trends and not absolute numbers for the purposes ofthe invention.

FIG. 46A-46C. List of all possible reduced pI variants created fromisotypic substitutions of IgG1-4. Shown are the pI values for the threeexpected species as well as the average delta pI between the heterodimerand the two homodimer species present when the variant heavy chain istransfected with IgG1-WT heavy chain.

FIG. 47. List of all possible increased pI variants created fromisotypic substitutions of IgG1-4. Shown are the pI values for the threeexpected species as well as the average delta pI between the heterodimerand the two homodimer species present when the variant heavy chain istransfected with IgG1-WT heavy chain.

FIG. 48A-48B. Matrix of possible combinations of first and secondmonomers for heterodimeric proteins of the invention, FcRn variants,Scaffolds, Fc variants and combinations, with each variant beingindependently and optionally combined from the appropriate source. Notethat the target antigens for the first and second monomer are eachindependently selected from the list provided in the first column.Legend: Legend A are suitable FcRn variants: 434A, 434S, 428L, 308F,259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y,252Y/254T/256E, 259I/308F/428L. Legend B are suitable scaffolds andinclude IgG1, IgG2, IgG3, IgG4, and IgG1/2. Sequences for such scaffoldscan be found for example in US Patent Publication No. 2012/0128663,published on May 24, 2012, which is hereby incorporated by reference inits entirety for all purposes and in particular for all teachings,figures and legends related to scaffolds and their sequences. Legend Care suitable Fc variants: 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D,267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 236R,328R, 236R/328R, 236N/267E, 243L, 298A and 299T. (Note, additionalsuitable Fc variants are found in FIG. 41 of US 2006/0024298, the figureand legend of which are hereby incorporated by reference in theirentirety). Legend D reflects the following possible combinations, again,with each variant being independently and optionally combined from theappropriate source Legend: 1) Monomer targets (each independentlyselected from the list in the first column) plus FcRn variants; 2)Monomer targets (each independently selected from the list in the firstcolumn) plus FcRn variants plus Scaffold; 3) Monomer targets (eachindependently selected from the list in the first column) plus FcRnvariants plus Scaffold plus Fc variants; 4) Monomer targets (eachindependently selected from the list in the first column) plus Scaffold5) Monomer targets (each independently selected from the list in thefirst column) plus Fc variants; 6) FcRn variants plus Scaffold; 7)Monomer targets (each independently selected from the list in the firstcolumn) plus Fc variants; 8) Scaffold plus Fc variants; 9) Monomertargets (each independently selected from the list in the first column)plus Scaffold plus Fc variants; and 10) Monomer targets (eachindependently selected from the list in the first column) plus FcRnvariants plus Fc variants.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS Overview of Invention

The present invention provides methods and compositions for modulating Tcells by administering heterodimeric proteins. Heterodimeric proteinsinclude without limitation heterodimeric antibodies (such as bispecificantibodies) and heterodimeric fusion proteins. By “modulating T cells”as discussed herein is meant suppressing or stimulating T cells. Ingeneral, the heterodimeric proteins of the invention are specific fortheir target T cell, meaning that the heterodimeric proteins primarilyaffect one type of T cell with little or no impact on other T celltypes. For example (and as is described in further detail herein),methods and compositions of the present invention for suppression orinduction of regulatory T cells (also referred to herein as “Tregs”)have little or no impact on other T cell types. Similarly, methods andcompositions of the invention for suppressing or inducing other T celltypes, such as cytotoxic T cells, primarily affect the cytotoxic T cellswith little or no impact on other T cell types.

By “suppressing T cells” as used herein refers to decreasing any aspectof T cell expression or function as compared to expression or functionin the absence of the administered heterodimeric protein. For example,suppression of regulatory T cells by admiinistering a heterodimericprotein includes suppression of the proliferation of regulatory T cellsas compared to proliferation in the absence of the administeredheterodimeric protein. “Inducing T cells” as used herein refers toincreasing any aspect of T cell expression or function as compared toexpression or function in the absence of the administered heterodimericprotein, including stimulation of the proliferation of the target Tcell.

As discussed in further detail herein, suppression or induction of Tcells can be measured with assays to quantify T cell numbers. Forexample, cell proliferation assays can be used to detect and quantitateT cells. Other methods of quantifying T cells, particularly Tregs, mayalso be used, including methods utilizing qPCR to measure the amount ofdemethylated FOXP3 (a Treg marker) that is present. Such assays aredescribed for example in Wieczorek et al., 2009, Cancer Res, 69(2):599-608, Vries et al., 2011, Clin Cancer Res, 17:841-848, and Baron etal., 2007, Eur. J. Immunol., 37:2378-2389, each of which is herebyincorporated by reference in its entirety for all purposes and inparticular for all teachings, figures and legends related to assays forFOXP3, demethylated FOXP3, and quantification of Tregs.

Suppression of T cells of a particular type is generally accomplished byadministering a heterodimeric protein that targets antigens specific forthat T cell type. For example, for regulatory T cells, administering aheterodimeric protein that targets both CD4 and CD25 reduces regulatoryT cell proliferation with minimal to no effect on other T cells, such asCD4+CD25− T effector cells or CD8+CD25+ cyotoxic T cells. Forsuppression of T cells, such a heterodimeric protein is generally abispecific antibody, although other multispecific antibodies and otherheterodimeric proteins such as fusion proteins are also contemplated. Incertain instances, suppression of regulatory T cells is accomplished byadministering a bispecific antibody that targets both CD4 and CD25; inother words, the bispecific antibody comprises two monomers in which onemonomer comprises an anti-CD4 binding domain and the other monomercomprises an anti-CD25 binding domain (“binding domain” and “bindingmoiety” are used interchangeably herein). “Anti-X binding domain” refersto a domain of the monomer that binds to X (i.e., an anti-CD25 bindingdomain is a part of the monomer that binds to CD25).

Other bispecific antibodies that suppress Tregs include withoutlimitation anti-CD25×anti-CTLA4, anti-CD25×anti-PD-1,anti-CD25×anti-CCR4, and anti-CD25×anti-G ITR antibodies.

Bispecific antibodies can also be used to suppress other T cell types,such as cytotoxic T cells. In some instances, one monomer of thebispecific antibody comprises an anti-CD8 domain, including withoutlimitation domains from anti-CD8 antibodies such as MCD8, 3B5, SK1,OKT-8, 51.1 and DK-25. The monomer with the anti-CD8 domain can becombined with a monomer comprising an anti-CD25 binding domain toproduce a bispecific antibody for suppression of cytoxic T cells.

Generally, the antigen binding domains of bispecific antibodies of theinvention are part of monomers that further comprise at least a heavychain constant region that contains a variant Fc domain as compared to aparent Fc domain.

In some situations, anti-CD4 and anti-CD8 targeting agents may furtherbe utilized in combination with T cell cytokines, including withoutlimitation IL-7, IL-12, IL-15, and IL-17.

Fc fusion proteins may also be used in accordance with the invention tosuppress T cells. For example, a fusion protein comprising an IL2protein on one arm can be engineered to have reduced ability to bind toIL2Rβ, IL2Rγ, and or IL2Rα in order to ablate IL2 receptor signaling.When coupled with an anti-CD4 antibody (or any other Treg surface markerantibody), this results in an anti-CD4×IL2 Fc-fusion capable ofsuppressing Treg cells through targeted binding to CD4 and CD25, butwithout the ability to induce Treg proliferation. In one non-limitingtheory, the mechanism of action for this fusion may be that it blocksendogenous IL2 from binding to receptor, thus preventing Tregproliferation. Exemplary embodiments of such fusion proteins areprovided in FIG. 23.

Heterodimeric proteins may also be used to induce T cells. As withmethods and compositions for suppressing T cells, induction of T cellsin accordance with the present invention is generally accomplished byadministering a heterodimeric protein that targets antigens and proteinsspecific for that T cell type. In specific instances, the presentinvention provides Fc fusion proteins comprising one monomer with abinding domain that targets a T cell marker and a second monomercomprising an IL2 protein. Examples of fusion proteins of use in thepresent invention for inducing T cells include without limitation fusionproteins that comprise IL2 on one monomer and one of the followingbinding domains on the other monomer: anti-CD4, anti-CCR4, anti-PD-1,anti-CD8, LAG3, and anti-CTLA4. In some situations, potency of thefusion proteins is increased by engineering the IL2 arm to increase theaffinity of IL2 for IL2Rα. Exemplary variants of IL2 of use in thepresent invention are listed in FIG. 23. Other cytokines that may beused in Fc fusion proteins of the invention include without limitationIL-7, IL-12, IL-15, and IL-17.

As will be appreciated and as is described in further detail herein, theheterodimeric proteins discussed herein may comprise a variety offormats, including those described herein (see for example FIGS. 3, 25,36 and 37) and those described in the art (see for example Kontermann etal., 2012, Landes Bioscience, which is incorporated herein by referencefor all purposes and in particular for all teachings related toheterodimeric proteins such as bispecific antibodies). In somesituations, bispecific antibodies may have one heavy chain containing asingle chain Fv (“scFv”, as defined herein) and the other heavy chain isa “regular” FAb format, comprising a variable heavy chain and a lightchain. This structure is sometimes referred to herein as “triple F”format (scFv-FAb-Fc) or the “bottle-opener” format, due to a roughvisual similarity to a bottle-opener, as described for example in U.S.Ser. No. 14/205,248, filed Mar. 11, 2014, which is hereby incorporatedby reference for all purposes and in particular for all teachingsrelated to the triple F or bottle opener format. In some situations,both of the heavy chains of the bispecific antibodies described hereincontain scFvs. Similarly, for any of the fusion proteins describedherein, the antibody arm of the fusion protein may be in the scFv orregular FAb format.

As is discussed in further herein, the heterodimeric proteins of thepresent invention may further include one or more amino acidsubstitutions in the Fc region that have the effect of increasing serumhalf-life, ablating binding to Fc□R, and/or increasing ADCC. Theheterodimeric proteins of the invention may also further include“heterodimerization variants” that, as is also described in furtherdetail herein, promote heterodimeric formation and/or allow for ease ofpurification of heterodimers over the homodimers. In certain situations,the heterodimeric proteins of the invention comprise one or more variantFc domains comprising an amino acid variant selected from among thevariants listed in FIGS. 33 and 34. In some situations, the amino acidvariants may further comprise variants selected from the group: 236R;239D; 239E; 243L; M252Y; V259I; 267D; 267E; 298A; V308F; 328F; 328R;330L; 332D; 332E; M428L; N434A; N434S; 236R/328R; 239D/332E; M428L;236R/328F; V259I/V308F; 267E/328F; M428L/N434S; Y436I/M428L;Y436V/M428L; Y436I/N434S; Y436V/N434S; 239D/332E/330L;M252Y/S254T/T256E; V259I/V308F/M428L; andE233P/L234V/L235A/G236del/S267K.

The methods and compositions of the present invention further includemethods for treating and/or alleviating the symptoms of diseases anddisorders affected by T cells, including without limitation cancer andautoimmune disease. In particular, methods and compositions of thepresent invention for the suppression of T cells, particularly Tregs,can be used to treat cancer. In addition, methods and compositions ofthe present invention for stimulation of T cells can be used to treatautoimmune disease.

As will be appreciated, suppression of T cells in accordance with thepresent invention may be used to treat any type of cancer. Bispecificantibodies targeting both CD4 and CD25 (or any other combination of Tcell markers as described herein and listed in FIG. 32) may in certainfurther embodiments be beneficial for the treatment of adult T cellleukemia (ATL), a rare disease associated with human T celllymphotrophic virus (HTLV-1). Diseased cells from ATL patients functionas regulatory cells and may arise from Treg cells, since these cellsdisplay a CD4⁺CD25⁺ phenotype consistent with that of Treg cells.Depletion of tumor cells with anti-CD4/CD25 bispecific antibodiescoupled to an enhanced effector function Fc domain may be a viabletreatment option.

As discussed above, the balance of Treg versus effector T cells can bedisregulated in autoimmune disease, and therapeutic approaches to favorhigher Treg ratios utilizing methods and compositions of the inventioncan be of use for treating such diseases. Induction and promotion of Tcells in accordance with the methods described herein can also be usedto treat (i.e., suppress) anti-graft responses in organ transplant andgraft-vs-host disease after allogeneic stem cell or bone marrowtransplant. Fc-fusion molecules, which in one non-limiting mechanism mayselectively ‘feed’ IL2 to Treg, promote the survival and expansion. Suchagents, which should alter the balance in favor of Treg vs effector Tcells, may provide a viable treatment option for controlling autoimmunedisease, organ transplant rejection, and graft-vs-host disease. Ingeneral, such treatments include the use of antibody-IL2 fusionproteins, in particular wherein a single IL2 protein is coupled with ananti-CD4 (or other Treg marker) moiety to provide selectivity for Tregversus effector T cells through the requirement for simultaneousengagement of CD4 and the high-affinity IL-2 receptor CD25.

Treatment of cancer, autoimmune disease or any other T cell associateddisease or disorder in accordance with the present invention generallyinvolves administering a composition containing a heterodimeric proteinof the invention (antibody or Fc fusion) to a patient in need thereof.

Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding are those shown in FIG. 7.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233# or E233( ) designatesa deletion of glutamic acid at position 233. Additionally, EDA233- orEDA233# designates a deletion of the sequence GluAspAla that begins atposition 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”, for example theIgG1/2 hybrid of FIG. 13. The protein variant sequence herein willpreferably possess at least about 80% identity with a parent proteinsequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Variant protein can referto the variant protein itself, compositions comprising the proteinvariant, or the DNA sequence that encodes it. Accordingly, by “antibodyvariant” or “variant antibody” as used herein is meant an antibody thatdiffers from a parent antibody by virtue of at least one amino acidmodification, “IgG variant” or “variant IgG” as used herein is meant anantibody that differs from a parent IgG (again, in many cases, from ahuman IgG sequence) by virtue of at least one amino acid modification,and “immunoglobulin variant” or “variant immunoglobulin” as used hereinis meant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification. “Fc variant” or “variant Fc” as used herein is meant aprotein comprising an amino acid modification in an Fc domain. The Fcvariants of the present invention are defined according to the aminoacid modifications that compose them. Thus, for example, N434S or 434Sis an Fc variant with the substitution serine at position 434 relativeto the parent Fc polypeptide, wherein the numbering is according to theEU index. Likewise, M428L/N434S defines an Fc variant with thesubstitutions M428L and N434S relative to the parent Fc polypeptide. Theidentity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as 428L/434S. It is noted that theorder in which substitutions are provided is arbitrary, that is to saythat, for example, 428L/434S is the same Fc variant as M428L/N434S, andso on. For all positions discussed in the present invention that relateto antibodies, unless otherwise noted, amino acid position numbering isaccording to the EU index. The EU index or EU index as in Kabat or EUnumbering scheme refers to the numbering of the EU antibody (Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporatedby reference.) The modification can be an addition, deletion, orsubstitution. Substitutions can include naturally occurring amino acidsand, in some cases, synthetic amino acids. Examples include U.S. Pat.No. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988A1; WO05/35727A2; WO 05/74524A2; J. W. Chin et al., (2002), Journal of theAmerican Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz,(2002), ChemBioChem 11:1135-1137; J. W. Chin, et al., (2002), PICASUnited States of America 99:11020-11024; and, L. Wang, & P. G. Schultz,(2002), Chem. 1-10, all entirely incorporated by reference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fvfragment” or “Fv region” as used herein is meant a polypeptide thatcomprises the VL and VH domains of a single antibody.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor, “FcγR” or “FcqammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life, are shown in paragraph [0320] ofthis specification.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as a bindingmoiety to a target protein, as described herein. In some cases, onemonomer of the heterodimeric protein comprises an antibody heavy chain(either including an scFv or further including a light chain) and theother monomer is a Fc fusion, comprising a variant Fc domain and aligand. In some embodiments, these “half antibody-half fusion proteins”are referred to as “Fusionbodies”.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. A wide number of suitable target antigens are described below.

By “strandedness” in the context of the monomers of the heterodimericproteins of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g. making the pI higher) then steric variants that are“charge pairs” that can be utilized as well do not interfere with the pIvariants, e.g. the charge variants that make a pI higher are put on thesame “strand” or “monomer” to preserve both functionalities.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V.kappa., V.lamda., and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10-4 M, at least about 10-5 M, at least about10-6 M, at least about 10-7 M, at least about 10-8 M, at least about10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, atleast about 10-12 M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

Methods and Compositions for Suppressing T Cells

In one aspect, the present invention provides methods and compositionsfor suppressing T cells. In preferred embodiments, the methods andcompositions for suppressing T cells are specific for one type of T cellwith limited to no impact on other T cells. In further embodiments, themethods and compositions of the present invention suppress Tregs withlimited to no impact on other T cell types. In other embodiments, themethods and compositions of the present invention suppress cytotoxic Tcells with limited to no impact on other T cell types.

In one aspect, the methods and compositions of the present inventionsuppress T cells by administration of heterodimeric proteins. Suchheterodimeric proteins include without limitation bispecific (althoughtrispecific, tetraspecific and higher order specificities are alsocontemplated) antibodies and fusion proteins.

In certain embodiments, suppression of T cells by methods andcompositions of the invention serve to increase the numbers and/orproliferation as compared to T cells that were not treated in accordancewith the present invention. In further embodiments, administration ofany of the heterodimeric proteins discussed herein serves to increasethe numbers and/or proliferation of T cells over that seen without theadministration of the heterodimeric protein (or that seen withadministration of a control protein) by at least 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%. In yet further embodiments,the increase in the is about 10-20%, 10-50%, 20-90%, 30-80%, 40-70%,50-60%. In further embodiments, an increase in numbers and/orproliferation is measured in comparison for the targeted T cell typeagainst the non-targeted type. For example, in embodiments in which theadministered heterodimeric protein suppresses regulatory T cells, theincrease in cell number and/or proliferation of regulatory T cells ismeasured in comparison to that of other T cell types. In still furtherembodiments, this comparative increase is at least 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% as compared to thenon-targeted T cell type. In yet further embodiments, the comparativeincrease in the targeted T cell type is about 10-20%, 10-50%, 20-90%,30-80%, 40-70%, 50-60% over that of the non-targeted T cell type.

In general, the heterodimeric proteins of use for suppression of T cellsin accordance with the present invention comprise two monomers, and eachmonomer comprises a heavy chain constant region with a variant Fc domainas compared to a parent Fc domain and an antigen binding moiety. Incertain embodiments, the variant Fc domain of one of the heavy chainconstant region of one of the monomers is different than the heavy chainconstant region of the other monomer.

In certain aspects, the heterodimeric proteins of the invention forsuppression of T cells comprise bispecific antibodies or Fc fusionproteins. The bispecific antibodies of the invention can take any formatdescribed herein and known in the art, including those pictured in FIG.3 and FIGS. 36 and 37. The antigen binding domains of these bispecificantibodies will generally comprise an anti-CD25 binding domain on onemonomer and a binding domain for a T cell marker on another arm. As willbe appreciated, however, any combination of proteins on T cells,including those listed in FIG. 32 can be targets in any combination forbispecific antibodies of the invention. In other words, bispecificantibodies of the invention for suppression of T cells may target anytwo T cell markers, including any two of those listed in FIG. 32.

In further exemplary embodiments, bispecific antibodies of the inventioncomprise an anti-CD25 binding domain on one monomer and an anti-CD4binding domain on the other monomer (such antibodies are also designatedherein as anti-CD25×anti-CD4 bispecific antibodies). In furtherembodiments, the bispecific antibodies of the invention comprise thefollowing combinations of antigen binding domains: anti-CD25×anti-CTLA4,anti-CD25×anti-PD-1, anti-CD25×anti-CCR4, Anti-CD4×Anti-CTLA4 andanti-CD4×Anti-CCR4 and anti-CD25×anti-GITR antibodies.

Treg cells express CD4 and CD25 simultaneously, and targeting bothantigens with a bispecific antibody could in one non-limiting theory bea powerful mechanism to selectively suppress Treg cells and allow theimmune system to mount a response against tumor cells. Thus, abispecific antibody allowing for simultaneous avid targeting of CD4 andCD25 (or any other combination of antigens as discussed above) may incertain embodiments reduce Treg cell proliferation, either via cytotoxicdepletion or by interfering with IL2-dependent proliferation. Such anapproach will in further embodiments have little or no effect onunactivated CD4+CD25⁻ T effector cells or CD8+CD25⁻ cytotoxic T cells.Although it may exhibit some suppression of activated CD4+CD25+ effectorT cells, Tregs are reported to have significantly higher dependence onIL-2 for survival (Malek and Bayer Nature 2004, hereby incorporated byreference in its entirety for all purposes and in particular for allteachings related to T cells), providing additional selectivity of thisapproach for Treg vs CD4 effector T cells.

Bispecific antibodies can also be used to suppress other T cell types,such as cytotoxic T cells. In some instances, one monomer of thebispecific antibody comprises an anti-CD8 domain, including withoutlimitation domains from anti-CD8 antibodies such as MCD8, 3B5, SK1,OKT-8, 51.1 and DK-25. The monomer with the anti-CD8 domain can becombined with a monomer comprising an anti-CD25 binding domain toproduce a bispecific antibody for suppression of cytoxic T cells.

Generally, the antigen binding domains of bispecific antibodies of theinvention are part of monomers that further comprise at least a heavychain constant region that contains a variant Fc domain as compared to aparent Fc domain.

Fc fusion proteins may also be used in accordance with the invention tosuppress T cells. For example, a fusion protein comprising an IL2protein on one arm can be engineered to have reduced ability to bind toIL2Rβ, IL2Rγ, and or IL2Rα in order to ablate IL2 receptor signaling.When coupled with an anti-CD4 antibody (or any other Treg surface markerantibody), this results in an anti-CD4×IL2 Fc-fusion capable ofsuppressing Treg cells through targeted binding to CD4 and CD25, butwithout the ability to induce Treg proliferation. In one non-limitingtheory, the mechanism of action for this fusion may be that it blocksendogenous IL2 from binding to receptor, thus preventing Tregproliferation. Exemplary embodiments of such fusion proteins areprovided in FIG. 23.

Any of the above described heterodimeric antibodies and fusion proteinsfor suppressing T cells may further include additional amino acidsubstitutions in the Fc domain. Such substitutions may include one orany combination of substitutions that affect heterodimer formation,serum half-life and/or binding to FcRn (also referred to herein as “Fcvariants”), binding to Fc receptors, or ADCC. Exemplary furthersubstitutions of use in any of the heterodimeric proteins discussedherein for suppression of T cells are listed in FIGS. 38-42 and 48.

Methods and Compositions for Inducing T Cells

In one aspect, the present invention provides methods and compositionsfor inducing T cells. In preferred embodiments, the methods andcompositions for inducing T cells are specific for one type of T cellwith limited to no impact on other T cells. In further embodiments, themethods and compositions of the present invention induce Tregs withlimited to no impact on other T cell types. In other embodiments, themethods and compositions of the present invention induce cytotoxic Tcells with limited to no impact on other T cell types.

“Inducing T cells” as used herein refers to increasing any aspect of Tcell expression or function as compared to expression or function in theabsence of the administered heterodimeric protein, including stimulationof the proliferation of the target T cell.

As discussed herein and understood in the art, induction (as well assuppression) of T cells can be measured with assays to quantify T cellnumbers. For example, cell proliferation assays can be used to detectand quantitate T cells. Other methods of quantifying T cells,particularly Tregs, may also be used, including methods utilizing qPCRto measure the amount of demethylated FOXP3 (a Treg marker) that ispresent. Such assays are described for example in Wieczorek et al.,2009, Cancer Res, 69(2): 599-608, Vries et al., 2011, Clin Cancer Res,17:841-848, and Baron et al., 2007, Eur. J. Immunol., 37:2378-2389, eachof which is hereby incorporated by reference in its entirety for allpurposes and in particular for all teachings, figures and legendsrelated to assays for FOXP3, demethylated FOXP3, and quantification ofTregs. Such assays can also be used to quantify the specificity ofinduction by providing quantitative measurements of numbers of T cellsof one type that are induced as compared to other types of T cells (forexample, numbers of Tregs induced as compared to cytotoxic T cells).

In certain embodiments, induction of T cells by methods and compositionsof the invention serve to increase the numbers and/or proliferation ascompared to T cells that were not treated in accordance with the presentinvention. In further embodiments, administration of any of theheterodimeric proteins discussed herein serves to increase the numbersand/or proliferation of T cells over that seen without theadministration of the heterodimeric protein (or that seen withadministration of a control protein) by at least 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%. In yet further embodiments,the increase in the is about 10-20%, 10-50%, 20-90%, 30-80%, 40-70%,50-60%. In further embodiments, an increase in numbers and/orproliferation is measured in comparison for the targeted T cell typeagainst the non-targeted type. For example, in embodiments in which theadministered heterodimeric protein induces regulatory T cells, theincrease in cell number and/or proliferation of regulatory T cells ismeasured in comparison to that of other T cell types. In still furtherembodiments, this comparative increase is at least 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% as compared to thenon-targeted T cell type. In yet further embodiments, the comparativeincrease in the targeted T cell type is about 10-20%, 10-50%, 20-90%,30-80%, 40-70%, 50-60% over that of the non-targeted T cell type.

In further aspects, induction of T cells is accomplished in accordancewith the present invention using heterodimeric Fc fusion proteins. Suchfusion proteins are also referred to herein as “fusionbodies” becausethey generally comprise two monomers, in which one monomer is an Fcdomain fused to a ligand, such as IL2, and the other monomer is a FAbmonomer comprising a heavy chain and a light chain.

In certain embodiments, Fc fusion proteins of the invention include onemonomer that comprises a T cell protein, including without limitationthose proteins listed in FIG. 32. In further embodiments, the monomercomprises all or a portion of an IL2 protein. As will be appreciated,the IL2 protein may comprise an IL2 protein from any source, includingany mammalian species. In preferred embodiments, the IL2 portion of themonomer comprises a sequence from human IL2. Variants of IL2 may also beused in Fc fusion proteins of the invention, including withoutlimitation variants such as those listed in FIG. 23.

In further embodiments, the Fc fusion proteins comprise a second monomerthat comprises a T cell protein, including without limitation any of theproteins listed in FIG. 32. In exemplary embodiments, the fusionbodiesof the present invention for induction of T cells have one monomer thatis an Fc domain fused to all or part of an IL2 protein and the secondmonomer comprises an antigen binding domain that targets one of thefollowing: CD4, CD8, CTLA-4, CCR4, and PD-1. In still furtherembodiments, the second monomer comprises both a heavy chain and a lightchain sequence, and the variable domains of those heavy and light chainsequences form the antigen-binding domain.

Methods of Making Compositions of the Invention

Any of the heterodimeric proteins discussed herein, including bispecificantibodies and heterodimeric Fc fusion proteins, can be made usingmethods known in the art and methods described in further detail herein.

In certain aspects, the invention provides one or more nucleic acidsencoding a composition according to any of the compositions describedherein. As will be appreciated, different monomers of the heterodimericproteins of the invention may be expressed using nucleic acids encodingall or a portion of one or more of the monomers of the protein. Thus,for example, for a bispecific antibody in which one monomer targets CD4and the other monomer target CD25, the present invention furtherprovides a nucleic acid encoding the first and second monomers asseparate molecules that are then assembled together by co-expression inthe same host cell. In other embodiments, the two monomers may beencoded in the same nucleic acid, in some embodiments within the samevector. In embodiments in which one or both of the monomers compriseboth heavy and light chain sequences, those sequences may also beencoded by one or by multiple nucleic acids.

In further embodiments, the invention further provides host cellsexpressing the one or more nucleic acids encoding the one or moremonomers of heterodimeric proteins of the invention. As will beappreciated and as is discussed above, the heterodimeric proteins of thepresent invention may be encoded by one or more nucleic acids. These oneor more nucleic acids may be expressed in a single host cell or inseparate host cells. For example, for heterodimeric proteins that are inthe bottle-opener format in which one of the monomers is an scFv and theother monomer is a FAb, there may be three nucleic acids encoding thisprotein: one for the scFv, one for the heavy chain sequence of the FAb,and one for the light chain sequence of the FAb. These three nucleicacids will in general be expressed in the same host cell in order toproduce the heterodimeric protein, although expression in separate hostcells is also contemplated.

In yet further embodiments, and in accordance with any of the above, thepresent invention provides a method of making any of the compositionsdescribed herein, the method including the step of culturing a host cellor more nucleic acids encoding a heterodimeric protein of the invention,including any of the bispecific antibodies or Fc fusion proteinsdescribed herein.

In further aspects, the present invention provides a method of purifyinga heterodimeric protein or bispecific antibody in accordance with any ofthe above, the method including: (a) providing a composition inaccordance with any of the heterodimeric proteins described herein, (b)loading the composition onto an ion exchange column; and (c) collectinga fraction containing the heterodimeric protein or bispecific antibody,thus purifying the protein or antibody.

Heterodimeric Proteins Overview

The present invention is directed to methods of modulating T cells usingnovel constructs to provide heterodimeric proteins that allow binding tomore than one antigen or ligand, e.g. to allow for multispecificbinding. The heterodimeric protein constructs are based on theself-assembling nature of the two Fc domains of the heavy chains ofantibodies, e.g. two “monomers” that assemble into a “dimer”.Heterodimeric proteins are made by altering the amino acid sequence ofeach monomer as more fully discussed below. Thus, the present inventionis generally directed to the creation of heterodimeric proteinsincluding antibodies, which can co-engage antigens in several ways,relying on amino acid variants in the constant regions that aredifferent on each chain to promote heterodimeric formation and/or allowfor ease of purification of heterodimers over the homodimers. Asdiscussed more fully below, the heterodimeric proteins can be antibodyvariants or based on Fc fusion proteins. Although much of the followingdiscussion is in terms of heterodimeric antibodies, it will beappreciated by those in the art and more fully described below, thediscussion applies equally to heterodimeric proteins that are based onFc fusion proteins (also referred to herein as fusionbodies).

Thus, the present invention provides bispecific antibodies (or, asdiscussed below, trispecific or tetraspecific antibodies can also bemade). An ongoing problem in antibody technologies is the desire for“bispecific” (and/or multispecific) antibodies that bind to two (ormore) different antigens simultaneously, in general thus allowing thedifferent antigens to be brought into proximity and resulting in newfunctionalities and new therapies. In general, these antibodies are madeby including genes for each heavy and light chain into the host cells.This generally results in the formation of the desired heterodimer(A-B), as well as the two homodimers (A-A and B-B). However, a majorobstacle in the formation of multispecific antibodies is the difficultyin purifying the heterodimeric antibodies away from the homodimericantibodies and/or biasing the formation of the heterodimer over theformation of the homodimers.

There are a number of mechanisms that can be used to generate theheterodimers of the present invention. In addition, as will beappreciated by those in the art, these mechanisms can be combined toensure high heterodimerization. Thus, amino acid variants that lead tothe production of heterodimers are referred to as “heterodimerizationvariants”. As discussed below, heterodimerization variants can includesteric variants (e.g. the “knobs and holes” or “skew” variants describedbelow and the “charge pairs” variants described below) as well as “pIvariants”, which allows purification of homodimers away fromheterodimers.

One mechanism is generally referred to in the art as “knobs and holes”(“KIH”) or sometimes herein as “skew” variants, referring to amino acidengineering that creates steric influences to favor heterodimericformation and disfavor homodimeric formation can also optionally beused; this is sometimes referred to as “knobs and holes”; as describedin U.S. Ser. No. 61/596,846 and U.S. Ser. No. 12/875,0015, Ridgway etal., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol.1997 270:26; U.S. Pat. No. 8,216,805, US 2012/0149876, all of which arehereby incorporated by reference in their entirety. The Figures identifya number of “monomer A-monomer B” pairs that include “knobs and holes”amino acid substitutions. In addition, as described in Merchant et al.,Nature Biotech. 16:677 (1998), these “knobs and hole” mutations can becombined with disulfide bonds to skew formation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” or “charge pairs”as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010),hereby incorporated by reference in its entirety. This is sometimesreferred to herein as “charge pairs”. In this embodiment, electrostaticsare used to skew the formation towards heterodimerization. As those inthe art will appreciate, these may also have an effect on pI, and thuson purification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R andothers shown in the Figures.

In the present invention, in some embodiments, pI variants are used toalter the pI of one or both of the monomers and thus allowing theisoelectric purification of A-A, A-B and B-B dimeric proteins.

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric proteins; one relies on the useof pI variants, such that each monomer has a different pI, thus allowingthe isoelectric purification of A-A, A-B and B-B dimeric proteins.Alternatively, some scaffold formats, such as the “triple F” format,also allows separation on the basis of size. As is further outlinedbelow, it is also possible to “skew” the formation of heterodimers overhomodimers. Thus, a combination of steric heterodimerization variantsand pI or charge pair variants find particular use in the invention.Additionally, as more fully outlined below, scaffolds that utilizescFv(s) such as the Triple F format can include charged scFv linkers(either positive or negative), that give a further pI boost forpurification purposes. As will be appreciated by those in the art, someTriple F formats are useful with just charged scFv linkers and noadditional pI adjustments, although the invention does provide the useof skew variants with charged scFv linkers as well (and combinations ofFc, FcRn and KO variants).

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B change be changed, with the pI of monomer A increasing and the pIof monomer B decreasing. As is outlined more fully below, the pI changesof either or both monomers can be done by removing or adding a chargedresidue (e.g. a neutral amino acid is replaced by a positively ornegatively charged amino acid residue, e.g. glycine to glutamic acid),changing a charged residue from positive or negative to the oppositecharge (aspartic acid to lysine) or changing a charged residue to aneutral residue (e.g. loss of a charge; lysine to serine.). A number ofthese variants are shown in the Figures.

Accordingly, in this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseits pI (wt A-+B or wt A−−B), or by increasing one region and decreasingthe other region (A+−B− or A−B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions of antibodiesthat are directed to altering the isoelectric point (pI) of at leastone, if not both, of the monomers of a dimeric protein to form “pIheterodimers” (when the protein is an antibody, these are referred to as“pI antibodies”) by incorporating amino acid substitutions (“pIvariants” or “pI substitutions”) into one or both of the monomers. Asshown herein, the separation of the heterodimers from the two homodimerscan be accomplished if the pIs of the two monomers differ by as littleas 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use inthe present invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the scFv and Fab of interest. Thatis, to determine which monomer to engineer or in which “direction” (e.g.more positive or more negative), the Fv sequences of the two targetantigens are calculated and a decision is made from there. As is knownin the art, different Fvs will have different starting pIs which areexploited in the present invention. In general, as outlined herein, thepIs are engineered to result in a total pI difference of each monomer ofat least about 0.1 logs, with 0.2 to 0.5 being preferred as outlinedherein.

Furthermore, as will be appreciated by those in the art and outlinedherein, heterodimers can be separated from homodimers on the basis ofsize. For example, as shown in FIGS. 36 and 37, heterodimers with twoscFvs can be separated by those of the “triple F” format and abispecific mAb. This can be further exploited in higher valency withadditional antigen binding sites being utilized. For example, asadditionally shown, one monomer will have two Fab fragments and theother will have one scFv, resulting in a differential in size and thusmolecular weight.

In addition, as will be appreciated by those in the art and outlinedherein, the format outlined herein can be expanded to providetrispecific and tetraspecific antibodies as well. In this embodiment,some variations of which are depicted in the FIG. 36A36M, it will berecognized that it is possible that some antigens are bound divalently(e.g. two antigen binding sites to a single antigen; for example, A andB could be part of a typical bivalent association and C and D can beoptionally present and optionally the same or different). As will beappreciated, any combination of Fab and scFvs can be utilized to achievethe desired result and combinations.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of the heavy chain(s), a more modularapproach to designing and purifying multispecific proteins, includingantibodies, is provided. Thus, in some embodiments, heterodimerizationvariants (including skew and purification heterodimerization variants)are not included in the variable regions, such that each individualantibody must be engineered. In addition, in some embodiments, thepossibility of immunogenicity resulting from the pI variants issignificantly reduced by importing pI variants from different IgGisotypes such that pI is changed without introducing significantimmunogenicity. Thus, an additional problem to be solved is theelucidation of low pI constant domains with high human sequence content,e.g. the minimization or avoidance of non-human residues at anyparticular position.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of bispecific antibodies, the ability toeliminate, minimize and/or distinguish when homodimers are present issignificant. Similarly, the ability to reliably test the reproducibilityof the heterodimeric protein production is important.

In addition to all or part of a variant heavy constant domain, one orboth of the monomers may contain one or two fusion partners, such thatthe heterodimers form multivalent proteins. As is generally depicted inthe Figures, and specifically FIG. 36A, the fusion partners are depictedas A, B, C and D, with all combinations possible. In general, A, B, Cand D are selected such that the heterodimer is at least bispecific orbivalent in its ability to interact with additional proteins.

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention cantake on a wide variety of configurations, as are generally depicted inFIGS. 36 and 37. Some figures depict “single ended” configurations,where there is one type of specificity on one “arm” of the molecule anda different specificity on the other “arm”. Other figures depict “dualended” configurations, where there is at least one type of specificityat the “top” of the molecule and one or more different specificities atthe “bottom” of the molecule. Furthermore as is shown, these twoconfigurations can be combined, where there can be triple or quadruplespecificities based on the particular combination. Thus, the presentinvention provides “multispecific” binding proteins, includingmultispecific antibodies. Thus, the present invention is directed tonovel immunoglobulin compositions that co-engage at least a first and asecond antigen. First and second antigens of the invention are hereinreferred to as antigen-1 and antigen-2 respectively.

One heterodimeric scaffold that finds particular use in the presentinvention is the “triple F” or “bottle opener” scaffold format. In thisembodiment, one heavy chain of the antibody contains an single chain Fv(“scFv”, as defined below) and the other heavy chain is a “regular” FAbformat, comprising a variable heavy chain and a light chain. Thisstructure is sometimes referred to herein as “triple F” format(scFv-FAb-Fc) or the “bottle-opener” format, due to a rough visualsimilarity to a bottle-opener (see FIG. 36B). The two chains are broughttogether by the use of amino acid variants in the constant regions (e.g.the Fc domain and/or the hinge region) that promote the formation ofheterodimeric antibodies as is described more fully below.

There are several distinct advantages to the present “triple F” format.As is known in the art, antibody analogs relying on two scFv constructsoften have stability and aggregation problems, which can be alleviatedin the present invention by the addition of a “regular” heavy and lightchain pairing. In addition, as opposed to formats that rely on two heavychains and two light chains, there is no issue with the incorrectpairing of heavy and light chains (e.g. heavy 1 pairing with light 2,etc.)

In addition to all or part of a variant heavy constant domain, one orboth of the monomers may contain one or two fusion partners, such thatthe heterodimers form multivalent proteins. As is generally depicted inthe FIG. 64 of U.S. Ser. No. 13/648,951, hereby incorporated byreference with its accompanying legend, the fusion partners are depictedas A, B, C and D, with all combinations possible. In general, A, B, Cand D are selected such that the heterodimer is at least bispecific orbivalent in its ability to interact with additional proteins. In thecontext of the present “triple F” format, generally A and B are an scFvand a Fv (as will be appreciated, either monomer can contain the scFvand the other the Fv/Fab) and then optionally one or two additionalfusion partners.

Furthermore, as outlined herein, additional amino acid variants may beintroduced into the bispecific antibodies of the invention, to addadditional functionalities. For example, amino acid changes within theFc region can be added (either to one monomer or both) to facilitateincreased ADCC or CDC (e.g. altered binding to Fcγ receptors); to allowor increase yield of the addition of toxins and drugs (e.g. for ADC), aswell as to increase binding to FcRn and/or increase serum half-life ofthe resulting molecules. As is further described herein and as will beappreciated by those in the art, any and all of the variants outlinedherein can be optionally and independently combined with other variants.

Similarly, another category of functional variants are “Fcγ ablationvariants” or “Fc knock out (FcKO or KO) variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g. FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific antibodies of theinvention, it is generally desirable to ablate FcγRIIIa binding toeliminate or significantly reduce ADCC activity.

Antibodies

The present invention relates to the generation of multispecificantibodies, generally therapeutic antibodies. As is discussed below, theterm “antibody” is used generally. Antibodies that find use in thepresent invention can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described below. In general, the term “antibody”includes any polypeptide that includes at least one constant domain,including, but not limited to, CH1, CH2, CH3 and CL.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Itshould be understood that therapeutic antibodies can also comprisehybrids of isotypes and/or subclasses. For example, as shown in USPublication 2009/0163699, incorporated by reference, the presentinvention covers pI engineering of IgG1/G2 hybrids.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g, Kabat et al., supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

It should be noted that for the IgG sequences depicted herein start atthe CH1 region, position 118; the variable regions are not includedexcept as noted. For example, the first amino acid, while designated asposition “1” in the sequence listing, corresponds to position 118 of theCH1 region, according to EU numbering.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or CK).

Another region of interest for additional substitutions, outlined below,is the Fc region. By “Fc” or “Fc region” or “Fc domain” as used hereinis meant the polypeptide comprising the constant region of an antibodyexcluding the first constant region immunoglobulin domain and in somecases, part of the hinge. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM, Fc may include the Jchain. For IgG, the Fc domain comprises immunoglobulin domains Cγ2 andCγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2(Cγ2). Although the boundaries of the Fc region may vary, the human IgGheavy chain Fc region is usually defined to include residues C226 orP230 to its carboxyl-terminus, wherein the numbering is according to theEU index as in Kabat. In some embodiments, as is more fully describedbelow, amino acid modifications are made to the Fc region, for exampleto alter binding to one or more FcγR receptors or to the FcRn receptor.

Accordingly, in some embodiments the present invention providesheterodimeric antibodies that rely on the use of two different heavychain variant Fc domains that will self-assemble to form heterodimericantibodies.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein, particularly inthe Fc domains to allow either heterodimerization formation or thepurification of heterodimers away from homodimers. A full lengthheterodimeric antibody is two heavy chains with different Fc domains andeither two light chains or a common light chain.

Alternatively, the antibodies can include a variety of structures as aregenerally shown in the Figures, including, but not limited to, antibodyfragments, monoclonal antibodies, bispecific antibodies, minibodies,domain antibodies, synthetic antibodies (sometimes referred to herein as“antibody mimetics”), chimeric antibodies, humanized antibodies,antibody fusions (sometimes referred to as “antibody conjugates”), andfragments of each, respectively.

In one embodiment, the antibody is an antibody fragment, as long as itcontains at least one constant domain which can be engineered to produceheterodimers, such as pI engineering. Other antibody fragments that canbe used include fragments that contain one or more of the CH1, CH2, CH3,hinge and CL domains of the invention that have been pI engineered. Forexample, Fc fusions are fusions of the Fc region (CH2 and CH3,optionally with the hinge region) fused to another protein. A number ofFc fusions are known the art and can be improved by the addition of theheterodimerization variants of the invention. In the present case,antibody fusions can be made comprising CH1; CH1, CH2 and CH3; CH2; CH3;CH2 and CH3; CH1 and CH3, any or all of which can be made optionallywith the hinge region, utilizing any combination of heterodimerizationvariants described herein.

scFv Embodiments

In some embodiments of the present invention, one monomer comprises aheavy chain comprises a scFV linked to an Fc domain, and the othermonomer comprises a heavy chain comprising a Fab linked to an Fc domain,e.g. a “typical” heavy chain, and a light chain. By “Fab” or “Fabregion” as used herein is meant the polypeptide that comprises the VH,CH1, VL, and CL immunoglobulin domains. Fab may refer to this region inisolation, or this region in the context of a full length antibody,antibody fragment or Fab fusion protein. By “Fv” or “Fv fragment” or “Fvregion” as used herein is meant a polypeptide that comprises the VL andVH domains of a single antibody.

Several of the heterodimeric antibody embodiments described herein relyon the use of one or more scFv domains, comprising the variable heavyand variable light chains, covalently linked using a linker, forming anantigen binding domain. Some embodiments herein use “standard” linkers,usually linkers of glycine and serine, as is well known in the art.

The present invention further provides charged scFv linkers, tofacilitate the separation in pI between a first and a second monomer.That is, by incorporating a charged scFv linker, either positive ornegative (or both, in the case of scaffolds that use scFvs on differentmonomers), this allows the monomer comprising the charged linker toalter the pI without making further changes in the Fc domains. Thesecharged linkers can be substituted into any scFv containing standardlinkers. Again, as will be appreciated by those in the art, charged scFvlinkers are used on the correct “strand” or monomer, according to thedesired changes in pI. For example, as discussed herein, to make tripleF format heterodimeric antibody, the original pI of the Fv region foreach of the desired antigen binding domains are calculated, and one ischosen to make an scFv, and depending on the pI, either positive ornegative linkers are chosen.

In addition, disulfide bonds can be engineered into the variable heavyand variable light chains to give additional stability.

Chimeric and Humanized Antibodies

In some embodiments, the antibody can be a mixture from differentspecies, e.g. a chimeric antibody and/or a humanized antibody. Ingeneral, both “chimeric antibodies” and “humanized antibodies” refer toantibodies that combine regions from more than one species. For example,“chimeric antibodies” traditionally comprise variable region(s) from amouse (or rat, in some cases) and the constant region(s) from a human.“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. Nos. 5,530,101;5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205; 5,821,337;6,054,297; 6,407,213, all entirely incorporated by reference). Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654, entirely incorporated by reference. A variety of techniquesand methods for humanizing and reshaping non-human antibodies are wellknown in the art (See Tsurushita & Vasquez, 2004, Humanization ofMonoclonal Antibodies, Molecular Biology of B Cells, 533-545, ElsevierScience (USA), and references cited therein, all entirely incorporatedby reference). Humanization methods include but are not limited tomethods described in Jones et al., 1986, Nature 321:522-525; Riechmannet al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33;He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, ProcNatl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, all entirelyincorporated by reference. Humanization or other methods of reducing theimmunogenicity of nonhuman antibody variable regions may includeresurfacing methods, as described for example in Roguska et al., 1994,Proc. Natl. Acad. Sci. USA 91:969-973, entirely incorporated byreference. In one embodiment, the parent antibody has been affinitymatured, as is known in the art. Structure-based methods may be employedfor humanization and affinity maturation, for example as described inU.S. Ser. No. 11/004,590. Selection based methods may be employed tohumanize and/or affinity mature antibody variable regions, including butnot limited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

Heterodimeric Heavy Chain Constant Regions

Accordingly, the present invention provides heterodimeric proteins basedon the use of monomers containing variant heavy chain constant regions,and specifically the Fc domains, as a first domain. By “monomer” hereinis meant one half of the heterodimeric protein. It should be noted thattraditional antibodies are actually tetrameric (two heavy chains and twolight chains). In the context of the present invention, one pair ofheavy-light chains (if applicable, e.g. if the monomer comprises an Fab)is considered a “monomer”. Similarly, a heavy chain region comprisingthe scFv is considered a monomer. In the case where an Fv region is onefusion partner (e.g. heavy and light variable domains) and anon-antibody protein is another fusion partner, each “half” isconsidered a monomer. Essentially, each monomer comprises sufficientheavy chain constant region to allow heterodimerization engineering,whether that be all the constant region, e.g. Ch1-hinge-CH2-CH3, the Fcregion (CH2-CH3), or just the CH3 domain.

The variant heavy chain constant regions can comprise all or part of theheavy chain constant region, including the full length construct,CH1-hinge-CH2-CH3, or portions thereof, including for example CH2-CH3 orCH3 alone. In addition, the heavy chain region of each monomer can bethe same backbone (CH1-hinge-CH2-CH3 or CH2-CH3) or different. N- andC-terminal truncations and additions are also included within thedefinition; for example, some pI variants include the addition ofcharged amino acids to the C-terminus of the heavy chain domain.

Thus, in general, one monomer of the present “triple F” construct is ascFv region-hinge-Fc domain) and the other is (VH-CH1-hinge-CH2-CH3 plusassociated light chain), with heterodimerization variants, includingsteric, isotypic, charge steering, and pI variants, Fc and FcRnvariants, ablation variants, and additional antigen binding domains(with optional linkers) included in these regions.

In addition to the heterodimerization variants (e.g. steric and pIvariants) outlined herein, the heavy chain regions may also containadditional amino acid substitutions, including changes for altering FcγRand FcRn binding as discussed below.

In addition, some monomers can utilize linkers between the variant heavychain constant region and the fusion partner. For the scFv portion ofthe “bottle-opener”, standard linkers as are known in the art can beused, or the charged scFv linkers described herein. In the case whereadditional fusion partners are made (e.g. FIGS. 1 and 2), traditionalpeptide linkers can be used, including flexible linkers of glycine andserine, or the charged linkers of FIG. 9. In some cases, the linkers foruse as components of the monomer are different from those defined belowfor the ADC constructs, and are in many embodiments not cleavablelinkers (such as those susceptible to proteases), although cleavablelinkers may find use in some embodiments.

The heterodimerization variants include a number of different types ofvariants, including, but not limited to, steric variants (includingcharge variants) and pI variants, that can be optionally andindependently combined with any other variants. In these embodiments, itis important to match “monomer A” with “monomer B”; that is, if aheterodimeric protein relies on both steric variants and pI variants,these need to be correctly matched to each monomer: e.g. the set ofsteric variants that work (1 set on monomer A, 1 set on monomer B) iscombined with pI variant sets (1 set on monomer A, 1 set on monomer B),such that the variants on each monomer are designed to achieve thedesired function, keeping in mind the pI “strandedness” such that stericvariants that may alter pI are put on the appropriate monomer.

It is important to note that the heterodimerization variants outlinedherein (for example, including but not limited to those variants shownin FIGS. 3 and 12), can be optionally and independently combined withany other variants, and on any other monomer. That is, what is importantfor the heterodimerization is that there are “sets” of variants, one setfor one monomer and one set for the other. Whether these are combinedfrom the FIGS. 1 to 1 (e.g. monomer 1 listings can go together) orswitched (monomer 1 pI variants with monomer 2 steric variants) isirrelevant. However, as noted herein, “strandedness” should be preservedwhen combinations are made as outlined above. Furthermore, for theadditional Fc variants (such as for FcγR binding, FcRn binding, etc.),either monomer, or both monomers, can include any of the listedvariants, independently and optionally. In some cases, both monomershave the additional variants and in some only one monomer has theadditional variants, or they can be combined.

Heterodimerization Variants

The present invention provides heterodimeric proteins, includingheterodimeric antibodies in a variety of formats, which utilizeheterodimeric variants to allow for heterodimeric formation and/orpurification away from homodimers.

Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are included in FIG.41A-41B.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. The Figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and hole” mutations can be combined with disulfide bonds to skewformation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which areincorporated expressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers, and can be independently and optionally included or excludedfrom the proteins of the invention.

pI (Isoelectric Point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Combinations of pI variants are shown in the figures.

Heavy Chain pI Changes

As outlined herein and shown in the figures, PI variants are shownrelative to IgG1, but all isotypes can be altered this way, as well asisotype hybrids. In the case where the heavy chain constant domain isfrom IgG2-4, R133E and R133Q can also be used.

Antibody Heterodimers Light Chain Variants

In the case of antibody based heterodimers, e.g. where at least one ofthe monomers comprises a light chain in addition to the heavy chaindomain, pI variants can also be made in the light chain. Amino acidsubstitutions for lowering the pI of the light chain include, but arenot limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E,K207E and adding peptide DEDE at the c-terminus of the light chain.Changes in this category based on the constant lambda light chaininclude one or more substitutions at R108Q, Q124E, K126Q, N138D, K145Tand Q199E. In addition, increasing the pI of the light chains can alsobe done.

Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. A number of these areshown in FIGS. 10A and 10B. That is, IgG1 is a common isotype fortherapeutic antibodies for a variety of reasons, including high effectorfunction. However, the heavy constant region of IgG1 has a higher pIthan that of IgG2 (8.10 versus 7.31). By introducing IgG2 residues atparticular positions into the IgG1 backbone, the pI of the resultingmonomer is lowered (or increased) and additionally exhibits longer serumhalf-life. For example, IgG1 has a glycine (pI 5.97) at position 137,and IgG2 has a glutamic acid (pI 3.22); importing the glutamic acid willaffect the pI of the resulting protein. As is described below, a numberof amino acid substitutions are generally required to significant affectthe pI of the variant antibody. However, it should be noted as discussedbelow that even changes in IgG2 molecules allow for increased serumhalf-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in FIGS. 46A-46C and 47. Asdiscussed herein, which monomer to engineer is generally decided by theinherent pI of the Fv and scaffold regions. Alternatively, the pI ofeach monomer can be compared.

Heterodimeric Fc Fusion Proteins

In addition to heterodimeric antibodies, the invention providesheterodimeric proteins that comprise a first monomer comprising avariant Fc region and a first fusion partner and a second monomer, alsocomprising a variant Fc region and a second fusion partner. The variantFc regions are engineered as herein for antibodies, and are thusdifferent, and in general the first and second fusion partners aredifferent as well. In some cases, where one monomer is antibody based(e.g. either comprising a standard heavy and light chain or a Fc domainwith an scFv) and the other is an Fc fusion protein, the resultingheterodimeric protein is called a “fusionbody”.

pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall′ Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half life as wild-type Fc (Dall′ Acqua etal. 2002, J. Immunol. 169:5171-5180, entirely incorporated byreference). The increased affinity of Fc for FcRn at pH 7.4 is thoughtto forbid the release of the Fc back into the blood. Therefore, the Fcmutations that will increase Fc's half-life in vivo will ideallyincrease FcRn binding at the lower pH while still allowing release of Fcat higher pH. The amino acid histidine changes its charge state in thepH range of 6.0 to 7.4. Therefore, it is not surprising to find Hisresidues at important positions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

pI variants that find use in this embodiment, as well as their use forpurification optimization, are disclosed in FIG. 20.

Combination of Heterodimeric Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants can be optionally and independently combinedin any way, as long as they retain their “strandedness” or “monomerpartition”. In addition, all of these variants can be combined into anyof the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

Nucleic Acids of the Invention

As discussed above regarding methods of making compositions of thepresent invention, the invention further provides nucleic acidcompositions encoding the heterodimeric proteins of the invention. Aswill be appreciated by those in the art, the nucleic acid compositionswill depend on the format and scaffold of the heterodimeric protein.Thus, for example, when the format requires three amino acid sequences,such as for the triple F format (e.g. a first amino acid monomercomprising an Fc domain and a scFv, a second amino acid monomercomprising a heavy chain and a light chain), three nucleic acidsequences can be incorporated into one or more expression vectors forexpression. Similarly, some formats (e.g. dual scFv formats such asdisclosed in FIG. 36M) only two nucleic acids are needed; again, theycan be put into one or two expression vectors.

Target Antigens

The heterodimeric proteins of the invention may target virtually anyantigens. The “triple F” format is particularly beneficial for targetingtwo (or more) distinct antigens. (As outlined herein, this targeting canbe any combination of monovalent and divalent binding, depending on theformat). Thus the immunoglobulins herein preferably co-engage two targetantigens, although in some cases, three or four antigens can bemonovalently engaged. Each monomer's specificity can be selected fromthe lists below. While the triple F immunoglobulins described herein areparticularly beneficial for targeting distinct antigens, in some casesit may be beneficial to target only one antigen. That is, each monomermay have specificity for the same antigen.

Particular suitable applications of the heterodimeric proteins hereinare co-target pairs for which it is beneficial or critical to engageeach target antigen monovalently. Such antigens may be, for example,immune receptors that are activated upon immune complexation. Cellularactivation of many immune receptors occurs only by cross-linking,achieved typically by antibody/antigen immune complexes, or via effectorcell to target cell engagement. For some immune receptors, activationonly upon engagement with co-engaged target is critical, as nonspecificcross-linking in a clinical setting can elicit a cytokine storm andtoxicity. Therapeutically, by engaging such antigens monovalently ratherthan multivalently, using the immunoglobulins herein, such activationoccurs only in response to cross-linking only in the microenvironment ofthe primary target antigen. The ability to target two different antigenswith different valencies is a novel and useful aspect of the presentinvention. Examples of target antigens for which it may betherapeutically beneficial or necessary to co-engage monovalentlyinclude but are not limited to immune activating receptors such as CD3,FcγRs, toll-like receptors (TLRs) such as TLR4 and TLR9, cytokine,chemokine, cytokine receptors, and chemokine receptors. In manyembodiments, one of the antigen binding sites binds to CD3, and in someembodiments it is the scFv-containing monomer.

Virtually any antigen may be targeted by the immunoglobulins herein,including but not limited to proteins, subunits, domains, motifs, and/orepitopes belonging to the following list of target antigens, whichincludes both soluble factors such as cytokines and membrane-boundfactors, including transmembrane receptors: 17-IA, 4-1BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE,ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, ActivinRIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAM9, ADAMTS,ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-1 (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3,-4, or -6, Neurturin, Neuronal growth factor (NGF), NGFR,NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM,OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR,PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE,PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF,PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA, prostatespecific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK,RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand,TL2), TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-α Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

Exemplary antigens that may be targeted specifically by theimmunoglobulins of the invention include but are not limited to: CD20,CD19, Her2, EGFR, EpCAM, CD3, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb(CD32b), FcγRI (CD64), Toll-like receptors (TLRs) such as TLR4 and TLR9,cytokines such as IL-2, IL-5, IL-13, IL-12, IL-23, and TNFα, cytokinereceptors such as IL-2R, chemokines, chemokine receptors, growth factorssuch as VEGF and HGF, and the like. To form the multispecific antibodiesof the invention, antibodies to any combination of these antigens can bemade; that is, each of these antigens can be optionally andindependently included or excluded from a multispecific antibodyaccording to the present invention.

The choice of suitable target antigens and co-targets depends on thedesired therapeutic application. Some targets that have provenespecially amenable to antibody therapy are those with signalingfunctions. Other therapeutic antibodies exert their effects by blockingsignaling of the receptor by inhibiting the binding between a receptorand its cognate ligand. Another mechanism of action of therapeuticantibodies is to cause receptor down regulation. Other antibodies do notwork by signaling through their target antigen. The choice of co-targetswill depend on the detailed biology underlying the pathology of theindication that is being treated.

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (Weiner et al., 2010, Nature Reviews Immunology10:317-327; Reichert et al., 2005, Nature Biotechnology 23[9]:1073-1078;herein expressly incorporated by reference). For anti-cancer treatmentit may be desirable to target one antigen (antigen-1) whose expressionis restricted to the cancerous cells while co-targeting a second antigen(antigen-2) that mediates some immunological killing activity. For othertreatments, it may be beneficial to co-target two antigens, for exampletwo angiogenic factors or two growth factors, that are each known toplay some role in proliferation of the tumor. Exemplary co-targets foroncology include but are not limited to HGF and VEGF, IGF-1R and VEGF,Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 andFcγRIIIa, CD20 and FcγRIIIa, Her2 and FcγRIIIa. An immunoglobulin of theinvention may be capable of binding VEGF and phosphatidylserine; VEGFand ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1;VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR andCDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20;CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 andCD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF andphosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and Her-2;c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20;IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 andVEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF;PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2;PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON andMSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR;VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4;VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4;CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 andCD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40;CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 andDR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 andCD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52;CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 andVEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, andEGFR and DLL4. Other target combinations include one or more members ofthe EGF/erb-2/erb-3 family.

Other targets (one or more) involved in oncological diseases that theimmunoglobulins herein may bind include, but are not limited to thoseselected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8,BMP6, IL12A, IL1A, IL1B, 1L2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1,FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2,FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,GRP, IGF1, IGF2, IL12A, IL1A, IL1B, 1L2, INHA, TGFA, TGFB1, TGFB2,TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2,CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4,CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3,IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL,TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1,IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR113,NR2F6, NR4A3, ESR1, ESR2, NROB1, NROB2, NR1D2, NR1H2, NR1H4, NR112,NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2,NR5A1, NR5A2, NR6 p1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10,FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2,IGFBP3, IGFBP6, IL12A, IL1A, IL1B, 1L2, IL24, INHA, INSL3, INSL4, KLK10,KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18,CDH19, CDH2O, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164,COL6A1, MTSS1, PAP, TGFB111, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1,CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3,FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A,IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID,PR1, PSCA, SLC2A2, SLC33 pI, SLC43 pI, STEAP, STEAP2, TPM1, TPM2, TRPC6,ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR,LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB 1, VEGF, VEGFC, ANGPTL3, BAI1,COL4A3, IL8, LAMA5, NRP1, NRP2, STAB 1, ANGPTL4, PECAM1, PF4, PROK2,SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2,EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2,TGFBR1, CCL2, CDH5, COL1A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B(p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin), CTSB(cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3,GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130),ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),NGFB (GF), NGFR, NME1 (M23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin),SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6(Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7(claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin),GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin),KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type IIkeratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2(COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1(mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2,THBS4, and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4,phophatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD80, CD55,CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL,VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2,NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1,PSMA, PSCA, SCD1, and CD59. To form the bispecific or trispecificantibodies of the invention, antibodies to any combination of theseantigens can be made; that is, each of these antigens can be optionallyand independently included or excluded from a multispecific antibodyaccording to the present invention.

Monoclonal antibody therapy has become an important therapeutic modalityfor treating autoimmune and inflammatory disorders (Chan & Carter, 2010,Nature Reviews Immunology 10:301-316; Reichert et al., 2005, NatureBiotechnology 23[9]:1073-1078; herein expressly incorporated byreference). Many proteins have been implicated in general autoimmune andinflammatory responses, and thus may be targeted by the immunoglobulinsof the invention. Autoimmune and inflammatory targets include but arenot limited to C5, CCL1 (1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15(MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2(mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b),CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11(1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5(ENA-78/LIX), CXCL6 (GCP-2), CXCL9, 1L13, IL8, CCL13 (mcp-4), CCR1,CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1(CCXCR1), IFNA2, IL10, 1L13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6,IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1(endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2,IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A,CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1,IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5,TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z,CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44,HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13,CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1,CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12,CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A,BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118,FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2,IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7,IL8, IL8RA, IL8RB, IL9, IL9R, IL10, MORA, IL10RB, IL11, IL12RA, IL12A,IL12B, IL12RB1, IL12RB2, 1L13, IL13RA1, IL13RA2, 1L15, IL15RA, IL16,1L17, IL17R, IL18, IL18R1, 1L19, IL20, KITLG, LEP, LTA, LTB, LTB4R,LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB111,TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A,TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4,TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

Exemplary co-targets for autoimmune and inflammatory disorders includebut are not limited to IL-1 and TNFalpha, IL-6 and TNFalpha, IL-6 andIL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13,IL-9 and IL-13, CD19 and FcγRIIb, and CD79 and FcγRIIb.

Immunoglobulins of the invention with specificity for the followingpairs of targets to treat inflammatory disease are contemplated: TNF andIL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF andalphaVbeta3; TNF and NGF; TNF and IL-23p19; TNF and IL-6; TNF and SOST;TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE andIL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 andIL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R andRANKL; IL-17A and IL-1beta; IL-1beta and RANKL; IL-1beta and VEGF; RANKLand CD-20; IL-1alpha and IL-1beta; IL-1alpha and IL-1beta.

Pairs of targets that the immunoglobulins described herein can bind andbe useful to treat asthma may be determined. In an embodiment, suchtargets include, but are not limited to, IL-13 and IL-1beta, sinceIL-1beta is also implicated in inflammatory response in asthma; IL-13and cytokines and chemokines that are involved in inflammation, such asIL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHRagonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13and ADAMS. The immunoglobulins herein may have specificity for one ormore targets involved in asthma selected from the group consisting ofCSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG,histamine and histamine receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6,IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17,IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1,IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCLi, CCL2, CCL3, CCL4, CCL5,CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1,CXCL1, CXCL2, CXCL3, XCLi, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STATE, TBX21, TGFB1, TNF,TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase.To form the bispecific or trispecific antibodies of the invention,antibodies to any combination of these antigens can be made; that is,each of these antigens can be optionally and independently included orexcluded from a multispecific antibody according to the presentinvention.

Pairs of targets involved in rheumatoid arthritis (RA) may beco-targeted by the invention, including but not limited to TNF andIL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1beta; TNF and MIF; TNFand IL-17; and TNF and IL-15.

Antigens that may be targeted in order to treat systemic lupuserythematosus (SLE) by the immunoglobulins herein include but are notlimited to CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2,IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A,HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, CD81, IFNB1, IL10, TNFRSF5,TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9,IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80,CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72,CD74, CD79A, CD79B, CR2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC,CHSTIO, HLA-A, HLA-DRA, and NT5E.; CTLA4, B7.1, B7.2, BlyS, BAFF, C5,IL-4, IL-6, IL-10, IFN-α, and TNF-α. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

The immunoglobulins herein may target antigens for the treatment ofmultiple sclerosis (MS), including but not limited to IL-12, TWEAK,IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200,IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An embodiment includesco-engagement of anti-IL-12 and TWEAK for the treatment of MS.

One aspect of the invention pertains to immunoglobulins capable ofbinding one or more targets involved in sepsis, in an embodiment twotargets, selected from the group consisting TNF, IL-1, MIF, IL-6, IL-8,IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissuefactor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFKB1, PROC,TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44,HMOX1, midkine, IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

In some cases, immunoglobulins herein may be directed against antigensfor the treatment of infectious diseases.

Antigen Binding Domains

As will be appreciated by those in the art, there are two basic types ofantigen binding domains, those that resemble antibody antigen bindingdomains (e.g. comprising a set of 6 CDRs) and those that can be ligandsor receptors, for example, that bind to targets without the use of CDRs.

Modified Antibodies

In addition to the modifications outlined above, other modifications canbe made. For example, the molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996, Nature Biotech. 14:1239-1245, entirelyincorporated by reference). In addition, there are a variety of covalentmodifications of antibodies that can be made as outlined below.

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesmay also be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

In addition, modifications at cysteines are particularly useful inantibody-drug conjugate (ADC) applications, further described below. Insome embodiments, the constant region of the antibodies can beengineered to contain one or more cysteines that are particularly “thiolreactive”, so as to allow more specific and controlled placement of thedrug moiety. See for example U.S. Pat. No. 7,521,541, incorporated byreference in its entirety herein.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125I or 131I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods, in addition to methods described below. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such ascynomolgusogen bromide-activated carbohydrates and the reactivesubstrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; and 4,330,440, all entirely incorporated byreference, are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983],entirely incorporated by reference), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

In addition, as will be appreciated by those in the art, labels(including fluorescent, enzymatic, magnetic, radioactive, etc. can allbe added to the antibodies (as well as the other compositions of theinvention).

Glycosylation

Another type of covalent modification is alterations in glycosylation.In another embodiment, the antibodies disclosed herein can be modifiedto include one or more engineered glycoforms. By “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to the antibody, wherein said carbohydrate composition differschemically from that of a parent antibody. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. A preferred form of engineered glycoformis afucosylation, which has been shown to be correlated to an increasein ADCC function, presumably through tighter binding to the FcγRIIIareceptor. In this context, “afucosylation” means that the majority ofthe antibody produced in the host cells is substantially devoid offucose, e.g. 90-95-98% of the generated antibodies do not haveappreciable fucose as a component of the carbohydrate moiety of theantibody (generally attached at N297 in the Fc region). Definedfunctionally, afucosylated antibodies generally exhibit at least a 50%or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umańa et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zurich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells, by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. For example,the “sugar engineered antibody” or “SEA technology” of Seattle Geneticsfunctions by adding modified saccharides that inhibit fucosylationduring production; see for example 20090317869, hereby incorporated byreference in its entirety. Engineered glycoform typically refers to thedifferent carbohydrate or oligosaccharide; thus an antibody can includean engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal. Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

Additional Fc Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγRreceptors, altered binding to FcRn receptors, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants and steric variants. Each set ofvariants can be independently and optionally included or excluded fromany particular heterodimeric protein.

FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors.Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto Fc□RIIIa generally results in increased ADCC (antibody dependentcell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances. Amino acid substitutions thatfind use in the present invention include those listed in U.S. Ser. No.11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287, 11/396,495,11/538,406, all of which are expressly incorporated herein by referencein their entirety and specifically for the variants disclosed therein.Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and299T.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 434A,428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,436V/428L and 259I/308F/428L.

Linkers

The present invention optionally provides linkers as needed, for examplein the addition of additional antigen binding sites, as depicted forexample in FIG. 2, where “the other end” of the molecule containsadditional antigen binding components. In addition, as outlined below,linkers are optionally also used in antibody drug conjugate (ADC)systems. When used to join the components of the central mAb-Fvconstructs, the linker is generally a polypeptide comprising two or moreamino acid residues joined by peptide bonds and are used to link one ormore of the components of the present invention. Such linkerpolypeptides are well known in the art (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123). A variety of linkers may find use in someembodiments described herein. As will be appreciated by those in theart, there are at least three different linker types used in the presentinvention.

“Linker” herein is also referred to as “linker sequence”, “spacer”,“tethering sequence” or grammatical equivalents thereof. Homo- orhetero-bifunctional linkers as are well known (see, 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated entirely by reference). A number of strategies may be usedto covalently link molecules together. These include, but are notlimited to polypeptide linkages between N- and C-termini of proteins orprotein domains, linkage via disulfide bonds, and linkage via chemicalcross-linking reagents. In one aspect of this embodiment, the linker isa peptide bond, generated by recombinant techniques or peptidesynthesis. The linker peptide may predominantly include the followingamino acid residues: Gly, Ser, Ala, or Thr. The linker peptide shouldhave a length that is adequate to link two molecules in such a way thatthey assume the correct conformation relative to one another so thatthey retain the desired activity. In one embodiment, the linker is fromabout 1 to 50 amino acids in length, preferably about 1 to 30 aminoacids in length. In one embodiment, linkers of 1 to 20 amino acids inlength may be used. Useful linkers include glycine-serine polymers,including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n isan integer of at least one, glycine-alanine polymers, alanine-serinepolymers, and other flexible linkers. Alternatively, a variety ofnonproteinaceous polymers, including but not limited to polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol, may find use as linkers,that is may find use as linkers.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cκ or Cλ Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g. TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

Antibody-Drug Conjugates

In some embodiments, the multispecific antibodies of the invention areconjugated with drugs to form antibody-drug conjugates (ADCs). Ingeneral, ADCs are used in oncology applications, where the use ofantibody-drug conjugates for the local delivery of cytotoxic orcytostatic agents allows for the targeted delivery of the drug moiety totumors, which can allow higher efficacy, lower toxicity, etc. Anoverview of this technology is provided in Ducry et al., BioconjugateChem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) andSenter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which arehereby incorporated by reference in their entirety.

Thus the invention provides multispecific antibodies conjugated todrugs. Generally, conjugation is done by covalent attachment to theantibody, as further described below, and generally relies on a linker,often a peptide linkage (which, as described below, may be designed tobe sensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides multispecific antibodies conjugated todrugs. As described below, the drug of the ADC can be any number ofagents, including but not limited to cytotoxic agents such aschemotherapeutic agents, growth inhibitory agents, toxins (for example,an enzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), or a radioactive isotope (that is, aradioconjugate) are provided. In other embodiments, the inventionfurther provides methods of using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of a multispecific antibody and one or more small moleculetoxins, such as a maytansinoids, dolastatins, auristatins, atrichothecene, calicheamicin, and CC1065, and the derivatives of thesetoxins that have toxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in 5,416,064, WO/01/24763,7,303,749, 7,601,354, U.S. Ser. No. 12/631,508, WO02/098883, 6,441,163,7,368,565, WO02/16368 and WO04/1033272, all of which are expresslyincorporated by reference in their entirety.

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises a multispecific antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (see U.S. Pat. No. 6,884,869expressly incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF (see US 2005/0238649,5,767,237 and 6,124,431, expressly incorporated by reference in theirentirety).

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. Nos. 5,635,483;5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettitet al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. PerkinTrans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin γ1 as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see 4,169,888, incorporated by reference) and duocarmycins aremembers of a family of antitumor antibiotics utilized in ADCs. Theseantibiotics appear to work through sequence-selectively alkylating DNAat the N3 of adenine in the minor groove, which initiates a cascade ofevents that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302,and 7,507,420, all of which are expressly incorporated by reference.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include a multispecific antibodyas the Antibody unit, a drug, and optionally a linker that joins thedrug and the binding agent.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with anmultispecific antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e.g. amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

ADC Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker; in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: 287)). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment,” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the multispecificantibodies of the invention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of 3H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of 3H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytotoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the multispecificantibody of the invention can be evaluated in a suitable animal model.For example, xenogenic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al., 1997,Nature Medicine 3: 402-408). Efficacy can be measured using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecificities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Thus for B cell tumors, the subject may experience a decrease in theso-called B symptoms, i.e., night sweats, fever, weight loss, and/orurticaria. For pre-malignant conditions, therapy with an multispecifictherapeutic agent may block and/or prolong the time before developmentof a related malignant condition, for example, development of multiplemyeloma in subjects suffering from monoclonal gammopathy of undeterminedsignificance (MGUS).

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the multispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an multispecific antibody used in the present invention is about0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as0.3, about 1, or about 3 mg/kg. In another embodiment, he antibody isadministered in a dose of 1 mg/kg or more, such as a dose of from 1 to20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the multispecific antibody is administered byinfusion in a weekly dosage of from 10 to 500 mg/kg such as of from 200to 400 mg/kg Such administration may be repeated, e.g., 1 to 8 times,such as 3 to 5 times. The administration may be performed by continuousinfusion over a period of from 2 to 24 hours, such as of from 2 to 12hours.

In one embodiment, the multispecific antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, ifrequired to reduce side effects including toxicity.

In one embodiment the multispecific antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the multispecific antibody.

In a further embodiment, the multispecific antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the multispecific antibody is administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In one embodiment, the multispecific antibody is administered by aregimen including one infusion of an multispecific antibody followed byan infusion of an multispecific antibody conjugated to a radioisotope.The regimen may be repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the multispecific antibody molecule thereof is usedin combination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-KBinhibitors, including inhibitors of IKB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,overexpressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below are for illustrative purposes only. Theseexamples are not meant to constrain any embodiment disclosed herein toany particular application or theory of operation.

Example 1. Constructing Anti-CD4×Anti-CD25 Bispecific Antibodies

A concept for suppressing Treg cells with anti-CD4×anti-CD25 bispecificswhile not affecting other T cell types is shown schematically in FIG. 1.

The ability of various anti-CD25 heavy chains to pair with anti-CD4light chains and anti-CD4 heavy chains to pair with anti-CD25 lightchains in order to create a “common light-chain” anti-CD4×CD25bispecific antibody was evaluated. Desired gene segments weresynthesized by Blue Heron Biotechnologies (Bothell, Wash.) fromsynthetic oligonucleotides and PCR products by automated gene synthesis.Antibody constructs in the pTT5 vector were expressed in 293E cells andpurified by standard Protein A, followed by IEX chromatography in orderto isolate the desired heterodimeric bispecific. Biacore was used toexamine binding of the various pairs to both CD4 and CD25 and theresults tabulated (FIG. 2). 100 nM of each variant was immobilized on aProtein A chip for 1 min, followed by flowing antigen (CD4 or CD25) at100 nM for 2 min dissociation. As can be seen from the data, theHuMax-TAC anti-CD25 heavy chain has the unique ability to pair with theanti-CD4 lights chains of OKT4A and zanolimumab, with theHuMax-TAC/OKT4A pair showing the strongest binding.

“Common light-chain” anti-CD4×CD25 bispecific antibodies wereconstructed by co-transfecting (in 293E cells) DNA encoding the heavychain of anti-CD4 antibody OKT4A_H0L0 with the heavy chain of anti-CD25antibody HuMAX-Tac and the light chain of anti-CD4 antibody OKTH0L0.These bispecific antibodies express well and have biophysical propertiesequivalent to normal monovalent IgG antibodies. Utilizing aheterodimeric Fc format, dual scFv-Fc anti-CD4×CD25 bispecificantibodies were also constructed and expressed. A third format, with anormal Fab-Fc on one side and scFv-Fc on the other side was alsoconstructed. Control “one-armed” antibodies were also constructed toevaluate the effects of monovalent antigen binding (e.g.Anti-CD4×empty-Fc or Anti-CD25 by empty-Fc). For all three formats,variants with different Fc regions were produced: IgG1, high ADCC(S239D/I332E), and Fc knockout (G236R/L328R or PVA_/S267K). Bispecificformats are shown schematically in FIG. 3. These bispecific antibodyvariants were evaluated for the ability to simultaneously bind both CD4and CD25 on Biacore. 100 nM of each variant was bound to a CD25 surface,followed by flowing of 100 nM of CD4 over the chip surface. An exampleof the data is shown in FIG. 4.

Although CD4 and CD25 antigens were initially targeted for suppressingTregs, other combinations of Treg markers may also be used in accordancewith the methods described herein, including combinations listed in FIG.32. Anti-CTLA4×Anti-CD25, Anti-PD-1×Anti-CD25, and Anti-CCR4×Anti-CD25bispecific antibodies were also constructed. Any of the formats shown inFIG. 3 can be made to bind to any combinations of the targets listed inFIG. 32.

Example 2. Suppression of Regulatory T Cells with Anti-CD4×Anti-CD25Bispecific Antibodies

Treg cells were generated in vitro using the following method. CD4⁺enriched T cells (isolated using the EasySep™ Human CD4⁺ T CellEnrichment Kit from Stemcell Technologies) from PBMC were incubated withanti-CD3/anti-CD28 beads (20 μl beads in 100 μl volume, or 4:1 beads tocell ratio using Dynabeads® Regulatory CD4⁺CD25⁺ T Cell Kit) with 500U/mL of IL2 in the presence of 0.1 μg/ml rapamycin for a week. Cellswere replaced with new culture with anti-CD3 (OKT3, eBiosciences) platebound at 0.5 μg/mL and soluble 0.5 μg/mL anti-CD28 (clone 28.2,eBiosciences) with 100 U/mL of IL2 and 0.1 μg/mL rapamycin.

Proliferation of Treg cells was assayed using CFSE cell proliferationassay or Alamar Blue cell viability assays in the presence of bispecificor control antibodies with 15 U/mL IL2. Results are shown in FIG. 5,FIG. 7, FIG. 12, FIG. 13, and FIG. 15. Anti-CD4×Anti-CD25 bispecifics11209 and 12143 (IgG1 and FcKO Fc, respectively) were able to suppressproliferation of Treg cells more strongly compared to anti-CD25 (6368)antibody alone, and no effect was seen with anti-CD4 mAb (10966) alone.These results demonstrate the increased suppression of Treg cells withavid targeting using anti-CD4×anti-CD25 bispecific antibodies. The Fv ofOKT4A was also humanized (OKT4A_H1L1) using the method of Lazar et al.,2007, Molecular Immunology, 44:1986-1998, hereby incorporated byreference in its entirety for all purposes and in particular for allteachings related to OKT4A, and this Fv was tested (FIG. 12 and FIG.15), along with bispecifics containing the alternative anti-CD4 FvIbalizumab (FIG. 13). The epitopes of OKT4A and Ibalizumab differ, withbinding of OKT4A to CD4 expected to block MHC II binding to CD4 whereasthe epitope of Ibalizumab is away from the MHC II binding site on CD4and its binding is not expected to be blocking. The precursor murine Fvof Ibalizumab (5A8) was also humanized to generate 5A8_H1L1. Bispecificswith the anti-CD4 Fv 5A8_H1L1 were also generated.

Example 3. Effect of Altering Antigen Binding Affinity ofAnti-CD4×Anti-CD25 Bispecific Antibodies

Variant bispecific antibodies and one-armed antibody controls wereconstructed in which the CD25 binding affinity was altered. TheAnti-TAC_H1.8L1 Fv (in 13531 and 13532) has 6-fold increased affinityfor CD25. Conversely, the Anti-TAC_H1L1.12 Fv (in 13533 and 13534) has17-fold lower CD25 affinity. These variants were assessed in cellproliferation assays (FIG. 15). A clear correlation between CD25affinity and potency can be seen. 13531 with increased CD25 affinity hasthe strongest inhibition of cell proliferation, while lower affinityresulted in a reduced effect on cell proliferation. A similar pattern isalso expected if CD4 affinity was altered. However, increasing theaffinity for CD4 may result in even greater potency on Tregs due to itslower expression level compared to CD25. This can be shown by lowerbinding of anti-CD4 mAbs on Tregs compared to anti-CD25 mAbs (shown inFIG. 8).

Example 4. Direct Binding of Anti-CD4×Anti-CD25 Bispecific Antibodies toTregs and Naïve CD4+ T Cells

Binding of Anti-CD4×anti-CD25 bispecifics and control antibodies wasmeasured to Tregs, naïve CD4+ T cells, and activated CD4+ and CD8+ Tcells. 200k Tregs were plated with antibodies at 4 μg/mL (4× serialdilutions, 8 total dilutions). Cells and Abs were incubated at 45 min onice and then washed and stained with secondary Ab anti-human F(ab)′2Fcgamma specific PE labeled at 1 μg/mL. Cells were washed and fixed with1% PFA overnight and data acquired on a FACS Canto II. Results are shownin FIGS. 8-11. Bispecifics and anti-CD25 mAbs bound more strongly toTregs compared to anti-CD4 mAbs, indicating that there may be a higherdensity of CD25 on Tregs compared to CD4. A clear avidity effect wasseen with the bispecifics. Direct binding to purified naive human CD4+ Tcells, activated CD4+, and activated CD8+ T cells was also assessed in asimilar manner. Results for these binding assays are shown in FIG. 14and FIGS. 16-22.

Example 5. Effect of Anti-CD4×Anti-CD25 Bispecific Antibodies on CellProliferation of CD4+CD25+(Helper T Cells) and CD8+CD25 (Cytotoxic TCells)

For suppression of Tregs, it is desirable to suppress Treg cells andhave little or no impact on other T cell types. To assess the impact ofAnti-CD4×Anti-CD25 bispecific antibodies on other T cell types, CFSElabeled PBMC were incubated with 12.5 ng/mL anti-CD3 and 15 U/mL IL2 for4 days in the presence of bispecific or control antibodies. Results areshown in FIG. 6 and FIG. 7. In this format, a clear dependence on FcγRbinding ability is seen. 11209 - Anti-CD4×Anti-CD25 IgG1 causessuppression of T-helper cells, while 12143 - Anti-CD4×Anti-CD25 FcKO hasa much reduced level of suppression. Anti-CD25 (6368) antibody alone isalso able to cause suppression of this T cell type, while anti-CD4 mAb(10966) alone shows limited activity (both are IgG1 Fc).

For cytotoxic T cells (CD8+CD25+), suppression was only seen with11209 - Anti-CD4×CD25 IgG1 and Anti-CD25 (6368). No suppression was seenwith 12143 - Anti-CD4×Anti-CD25 FcKO or anti-CD4 mAb (10966). Again, aclear dependence on FcγR binding ability of the bispecifics is seen.

Example 6. Constructing Bispecific Anti-CD4×IL2 Fc-Fusions

The concept of inducing Treg cells with anti-CD4×IL2 Fc-fusions whilenot affecting other T cell types is shown schematically in FIG. 24.

Anti-CD4×IL2 Fc-fusions were designed and constructed from the sequencesof human IL2 and the anti-CD4 antibody OKT4A (FIG. 25). Constructs inthe pTT5 vector were expressed in 293E cells and purified using ProteinA and IEX chromatography to isolate the desired heterodimeric Fc-fusion.SEC and SDS-PAGE analysis of the Protein A purified material as well asthe final IEX purified material are shown in FIG. 26. Anti-CD4×IL2Fc-fusions were homogeneous and obtained in high purity. All Fc-fusionswere expressed with a FcγR knocked out binding Fc region (IgG1G236R/L328R or IgG1 PVA_/S267K). Anti-CD4×IL2 Fc-fusions using theAnti-CD4 mAbs Ibalizumab and 5A8_H1L1 were also constructed, as wereAnti-CCR4×IL2, Anti-CTLA4×IL2, and Anti-PD1×IL2 antibody Fc-fusions.Bispecific IL2 Fc-fusions with antibodies against any of the Tregmarkers listed in Table 1 could also be constructed. Alternatively,similar variants may possess superior selectivity for Treg agonismversus other T cell types.

Example 7. Induction of Regulatory T Cells (Tregs) by Anti-CD4×IL2Fc-Fusions

Treg cells were generated in vitro using the following method. CD4⁺enriched T cells (isolated using the EasySep™ Human CD4+ T CellEnrichment Kit from Stemcell Technologies) from PBMC were incubated withanti-CD3/anti-CD28 beads (20 μl beads in 100 μl volume, or 4:1 beads tocell ratio using Dynabeads® Regulatory CD4⁺CD25⁺ T Cell Kit) with 500U/mL of IL2 in the presence of 0.1 μg/mL rapamycin for a week. Cellswere replaced with new culture with anti-CD3 (OKT3, eBiosciences) platebound at 0.5 μg/mL and soluble 0.5 μg/mL anti-CD28 (clone 28.2,eBiosciences) with 100 U/mL of IL2 and 0.1 μg/mL rapamycin.

Induction of Treg cells was assayed using the alamar blue cell viabilityassay in the presence of anti-CD4×IL2 Fc-fusions or control antibodies.Results are shown in FIG. 27. Increased viability of Treg cells was seenfor the anti-CD4×IL2 Fc-fusions as well as IL2-only Fc-fusions.Anti-CD25 and anti-CD4 control antibodies showed no induction. TheIL2-only Fc fusion (13044) served as a proxy for the reduced level ofinduction expected for cytotoxic T cells (CD8⁺CD25⁺).

Example 8. Suppression or Induction of Regulatory T Cells UsingAnti-CD4×1L2 Fc-Fusions Engineered for Reduced or Increased IL2-ReceptorSignaling

IL2 are engineered in order to alter the ratio of induction for Tregcells versus other types of IL2 receptor expressing cells (i.e. NKcells). For example, a dominant-negative IL2 Fc-fusion is created byengineering IL2 to have reduced ability to bind to IL2Rβ, IL2Rγ, and orIL2Rα in order to ablate IL2 receptor signaling. When coupled with ananti-CD4 antibody (or other Treg surface marker antibody), this resultsin an anti-CD4×IL2 Fc-fusion capable of suppressing Treg cells throughtargeted binding to CD4 and CD25, but without the ability to induce Tregproliferation. This Fc-fusion blocks endogenous IL2 from binding toreceptor.

Likewise, more potent Anti-CD4×IL2 Fc-fusions inducers are engineered byincreasing the affinity of IL2 for IL2Rα. Exemplary variants of IL2 ofuse in the present invention are listed in FIG. 23.

Example 9. Suppression and Induction of Cytotoxic T Cells withAnti-CD8×Anti-CD25 Bispecific Antibodies or Anti-CD8×IL2 Fc-Fusions

Anti-CD8 antibodies including MCD8, 3B5, SK1, OKT-8, 51.1 or DK-25 arecombined with an anti-CD25 antibody to make a bispecific antibody forsuppression of cytotoxic T cells. Alternatively, in order to inducecytotoxic T cells, an Fc-fusion consisting of IL2 combined with ananti-CD8 antibody are used. Avidity may also drive IL-2 activation bybinding the low affinity (beta/gamma) IL-2 receptor, circumventing therequirement for CD25, thus also being effective on non-activated CD8.Methods for suppression and induction are shown schematically in FIG. 28and FIG. 29. These approaches are useful for treating cancer orautoimmune diseases, respectively.

Example 10. Evaluation of Treg Suppressor and Inducer Variants in a GVHDMouse Model

Variants of the invention are evaluated in a Graft-versus-Host Diseasemodel conducted in NSG SCID mice such as those conducted in Mutis etal., Clin Cancer Res (12), 2006. When NSG SCID mice are injected withhuman PBMCs they develop an autoimmune response against the human PBMCs,and this has been shown to be Treg dependent. NSG SCID mice injectedwith human PBMCs and then treated with a Treg suppression bispecificantibody such as 12143, 12462, 13025, or 13529 will have an exacerbationof disease and will die more quickly compared to untreated mice.Conversely, mice can be given a Treg inducing bispecific IL2-Fc fusionsuch as 13027 and they have a less severe disease and live longer thanuntreated mice.

Example 11. Evaluation of Treg Suppressor Mouse Surrogate Variants inSyngeneic Mouse Tumor Models

Mouse surrogate bispecific antibodies and IL2-Fc fusions can be made andstudied in syngeneic mouse tumor models. The Treg suppressor bispecificAnti-mCD4×Anti-mCD25 can be made using the Anti-mouse CD4 antibody GK1.5and the Anti-mouse CD25 antibody PC61. Tumors can be introduced innormal mice and then the mice treated with surrogate bispecificantibody. Suppression of the mouse Tregs should allow the mousecytotoxic T cells to fight the tumor, resulting in a decreased tumorvolume.

Example 12. Evaluation of Treg Inducer Mouse Surrogate Variants in anEAE Mouse Model

Mouse surrogate Treg inducer IL2-Fc fusion bispecifics can be created byusing human IL2 with an anti-mouse CD4 antibody such as GK1.5. Human IL2is known to bind to the mouse IL2 receptor. Experimental autoimmuneencephalomyelitis (EAE) is a mouse model of autoimmunity. Mice can beinduced for EAE and then treated with mouse surrogate Anti-mCD4×IL2bispecific Fc-fusions. Induction of mouse Tregs should result in lesssevere disease.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments have been described above for purposes ofillustration, it will be appreciated by those skilled in the art thatnumerous variations of the details may be made without departing fromthe invention as described in the appended claims.

1.-69. (canceled)
 70. A heterodimeric protein comprising: (a) a firstmonomer comprising: (i) a first Fc domain; (ii) an IL-2 protein; and (b)a second monomer comprising: (i) a second Fc domain.
 71. Theheterodimeric protein according to claim 70, wherein the first andsecond Fc domains are variant Fc domains comprising amino acid variantsselected from the group consisting of: L368D/K370S and S364K;L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; and K370S andS364K/E357Q, wherein numbering is according to EU index as in Kabat. 72.The heterodimeric protein according to claim 71, wherein the firstand/or second variant Fc domain further comprises an amino acid variantindependently selected from the group consisting of: 236R, 328R, 330L,236R/328R, 239D/332E, E233P/L234V/L235A/G236del/S239K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G, E233P/L234V/L235A/G236del, andE233P/L234V/L235A/G236del/S267K, wherein numbering is according to EUindex as in Kabat.
 73. The heterodimeric protein according to claim 70,wherein the first Fc domain is a variant Fc domain comprising amino acidvariants E233P/L234V/L235A/G236del/S267K and S364K/E357Q, and whereinthe second Fc domain is a variant Fc domain comprising amino acidvariants E233P/L234V/L235A/G236del/S267K and L368D/K370S, whereinnumbering is according to EU index as in Kabat.
 74. The heterodimericprotein according to claim 70, wherein the IL-2 protein is an IL-2variant having reduced ability to bind to IL-2Rβ, IL-2Rγ, and/or IL-2Rα.75. The heterodimeric protein according to claim 70, wherein the IL-2protein is an IL-2 variant having increased ability to bind to IL-2Rα.76. The heterodimeric protein according to claim 70, wherein the IL-2protein is an IL-2 variant having reduced ability to bind to IL-2Rβand/or IL-2Rγ and increased ability to bind to IL-2Rα.
 77. A method ofinducing T cells, the method comprising contacting the T cells with acomposition comprising a heterodimeric protein according to claim 70.78. The method according to claim 77, wherein the T cells are regulatoryT cells (Tregs).
 79. A method of suppressing T cells, the methodcomprising contacting the T cells with a composition comprising aheterodimeric protein according to claim
 70. 80. A method A method oftreating an autoimmune disease in a subject, the method comprisingadministering to the subject a composition comprising a heterodimericprotein according to claim
 70. 81. A method A method of treating acancer in a subject, the method comprising administering to the subjecta composition comprising a heterodimeric protein according to claim 70.82. A nucleic acid composition encoding a heterodimeric protein, thenucleic acid composition comprising: a) a first nucleic acid encodingthe first monomer of claim 70; and b) a second nucleic acid encoding thesecond monomer of claim
 70. 83. A host cell comprising the nucleic acidcomposition of claim
 82. 84. A method of making a heterodimeric proteincomprising culturing a host cell according to claim 83 under conditionswhereby the heterodimeric protein is produced.
 85. A method of purifyinga heterodimeric protein according to claim 70, the method comprising:(a) providing a composition comprising the heterodimeric protein; (b)loading the composition onto an ion exchange column; and (c) collectinga fraction containing the heterodimeric protein.
 86. An IL-2 Fc fusioncomprising: (a) a first monomer comprising: (i) a first Fc domain; (ii)a first IL-2 protein; and (b) a second monomer comprising (i) a secondFc domain; and (ii) a second IL-2 protein.
 87. The IL-2 Fc fusionaccording to claim 86, wherein the first and/or second IL-2 protein isan IL-2 variant engineered to have reduced ability to bind to IL-2Rβ,IL-2Rγ, and/or IL-2Rα.
 88. A method of inducing T cells, the methodcomprising contacting the T cells with an IL-2 Fc fusion according toclaim
 86. 89. The method according to claim 88, wherein the T cells areregulatory T cells (Tregs).
 90. A method of suppressing T cells, themethod comprising contacting the T cells with an IL-2 Fc fusionaccording to claim
 86. 91. A method A method of treating an autoimmunedisease in a subject, the method comprising administering to the subjectan IL-2 Fc fusion according to claim
 86. 92. A method A method oftreating a cancer in a subject, the method comprising administering tothe subject an IL-2 Fc fusion according to claim
 86. 93. A nucleic acidcomposition encoding an IL-2 fusion, the nucleic acid compositioncomprising: a) a first nucleic acid encoding the first monomer of claim86; and b) a second nucleic acid encoding the second monomer of claim86.
 94. A host cell comprising the one or more nucleic acids of claim86.
 95. A method of making an IL-2 Fc fusion, the method comprisingculturing a host cell according to claim 94 under conditions whereby theIL-2 Fc fusion is produced.
 96. A method of purifying an IL-2 Fc fusionaccording to claim 86, the method comprising: (a) providing acomposition comprising the IL-2 Fc fusion; (b) loading the compositiononto an ion exchange column; and (c) collecting a fraction containingthe IL-2 Fc fusion.